Characteristics of Animals under Specific Environmental Conditions

### **Chapter 4**

Techniques of Using Peripheral Blood Mononuclear Cells as the Cellular System to Investigate How of the Bovine Species (Indian Zebu-Jersey Crossbreds) Responds to *in vitro* Thermal Stress Stimulation (Thermal Assault/Heat Shock)

*Gbolabo Olaitan Onasanya,*

*Aranganoor Kannan Thiruvenkadan, Alice Adishetu Yisa, Krishnaswamy Gopalan Tirumurugaan, Murali Nagarajan, Saravanan Ramasamy, Raja Angamuthu, George Mutani Msalya and Christian Obiora Ikeobi*

#### **Abstract**

Animal production is negatively impacted by global warming and is subject to serious consequences for livestock production systems. In order to understand how PBMCs of Indian Zebu-Jersey crossbreds respond to various levels and durations of thermal assault and heat shock, in this chapter we will discuss techniques involving *in vitro* thermal stress stimulation (TSS) to stimulate bovine peripheral blood mononuclear cells (PBMCs) under various thermal assault conditions (TACs), including normal to extreme temperatures and varying durations of thermal exposure (DTEs). The consequences of thermal stress on bovine species can be lessened and managed with an understanding of how PBMCs as a cellular system respond to in *vitro* heat shock and thermal assault. To learn more about how Indian Zebu-Jersey crossbreds respond to *in vitro* thermal conditions, it may also be possible to explore the relationship between the decrease in PBMCs count during *in vitro* TSS and the expression of the heat shock protein genes (HSPs) such as *HSPs* 70 and 90 genes. This will be exploited to discover how Indian Zebu-Jersey crossbreds respond *in vivo* to diverse environmental thermal conditions and will further enable *in vivo* understanding of the potential for thermotolerance in bovine species for better adaptability, survival, and production performance.

**Keywords:** heat shock, PBMCs, thermal assault, cellular system, Zebu Cattle, heat stress

#### **1. Introduction**

Livestock animals are required to raise their respiratory rate and peripheral blood flow in the tropics due to the harsh weather conditions, which has a detrimental effect on physiological and production performance, including poor milk quality [1]. Environmental thermal conditions have made it difficult for both humans and animals to survive in the face of the existential danger posed by climate change, which has had a variety of negative effects on performance, production, and food security [2]. Cattle as well as other animals can suffer severe effects from thermal stress (TS), including decreased feed intake, low milk production, stunted growth, poor health, decreased activity, and poor performance [3], they also succumb to hyperthermia if thermal assaults are not mitigated [3].

Furthermore, livestock animals are compelled to adapt and survive under assault of thermal conditions or extreme environmental conditions in the tropics, which has a negative impact on their physiology and production performance. The ability of cattle and other animals to maintain homeostasis is negatively impacted by changes in external temperatures and relative humidity, which forces them to actively maintain the internal body temperature (IBT) required for their survival and productivity [4]. Homeothermy is the ability of an animal to regulate its internal body temperature (IBT) in the face of thermal challenges from the environment [1]. TS is attained when an animal's BT is raised over its usual physiological range. The condition results in increased management expenses, lowers food security, and has a negative influence on income production, all of which create economic loss [5].

According to earlier research findings [6–9], variations in thermal assault conditions (TACs) and heat shock have an impact on cellular integrity, proliferation, and viability as well as RNA concentration, making animals more susceptible to opportunistic infections caused by weakened immune systems and a decline in productivity and reproduction [1]. Because RNA is a heat-labile nucleic acid, it is extremely unstable when exposed to harsh environmental conditions, especially heat or thermal assault and as such the degradation of RNA nucleotides and subsequent mutational damage to the structure of nucleic acids that affect nucleic acid synthesis and functions have also been linked to harsh environmental TACs [1]. Previous studies revealed that temperature variations had a significant impact on the production and proliferation of RNA nucleotides [1]. For instance, a moderate 37°C *in vitro* temperature mimics and resembles the BT of mammalian species and increases RNA synthesis and proliferation as well as cell survival [1, 8, 10]. In order for cattle to perform better in terms of production and reproduction abilities, it is necessary to mitigate the effects of harsh environmental conditions [3].

In order to obtain biological information about how cellular systems react to heat shock following exposure to TACs, Onasanya and his team [1] performed *in vitro* TSS of PBMCs on PBMCs of Indian Zebu-Jersey crossbreds maintained under stressful thermal conditions. In this chapter, the methods and techniques employed by the authors will be adequately discussed.

*Techniques of Using Peripheral Blood Mononuclear Cells as the Cellular System… DOI: http://dx.doi.org/10.5772/intechopen.109431*

#### **2. Blood sample collection and experimental animals**

Seventy (70) Indian Zebu-Jersey crossbred animals were between the age of four and six years. Blood samples (10 mL per animal) were taken aseptically in EDTA bottle (**Figure 1**). After blood collection, the samples were transported in cooled iced-packed box and PBMCs were isolated within two hours of collection [1]. The reason for this is that, immediately the blood is collected the cells will continue to survive on the blood glucose, once the blood glucose is exhausted the cells will begin to die and they won't be able to respond to *In vitro* TSS.

#### **3. Procedures for isolation of peripheral blood mononuclear cells**

Ten (10) mL of animal blood samples were homogenized and properly mixed. Then, homogenized blood was added in an equal V/V ratio to 10 mL of previously prepared phosphate-buffered saline (PBS) (HiMedia Laboratories, Mumbai, India). A gentle homogenization and thorough mixing with pipetting up and down followed. A new 50 mL conical tube containing 3 mL of Histopaque®-1077 (Sigma-Aldrich Co. LLC, Darmstadt, Germany) was then carefully filled with the blood-PBS mixture, and the tube was centrifuged at 400 g for 20 min in a REMI R-4C laboratory centrifuge (Goregaon East, Mumbai - 400 062, India) [1].

Using Histopaque®-1077 (Sigma-Aldrich Co. LLC), fresh whole blood was fractionated, and four separate layers were visible: the top layer was yellowish plasma, the bottom layer was milky PBMCs, and the top layer was histopaque. The top layer

**Figure 1.** *Showing India Zebu–Jersey crossbred cattle.*

#### **Figure 2.**

*(a) showing the hemocytometer's ability to detect peripheral blood mononuclear cells under a microscope; (b) shows the fractional separation of a blood sample, which reveals peripheral blood mononuclear cells as well as other fractional layers.*

contained erythrocytes and granulocytes (**Figure 2b**). **Figure 2a** show the detection of the PBMCs under microscope.

After centrifugation, PBMCs were collected with the least amount of plasma (and Histopaque®-1077) and transferred into a clean15 mL conical tube. The PBMC suspension was then extensively homogenized by pipetting up and down after adding 10 mL of PBS (PBS was used to wash the PBMCs), followed by 10 min of centrifugation at 100 g. After that, the supernatant was discarded in order to recover the PBMC pellet. The conical tube was then flicked until the PBMC pellets were fully resuspended in the remaining PBS solution. 10 mL of PBS solution was added to the pellet and thoroughly mixed by up-and-down pipetting. After centrifuging the mixture at 100 g for 10 min, the supernatant was discarded**.**

The method (washing with PBS, mixing, and centrifuging at 100 g for 10 min was performed three times) to recover the PBMCs pellet. The PBMCs were then resuspended in 1 mL of a mixture consisting of 100 mL of fetal bovine serum (FBS) and 900 mL of basic medium (RPMI-1640): 900 mL (HiMedia, Laboratories), and gently mixed by up and down pipetting in the FBS-RPMI mixture. Trypan blue dye exclusion method was used to count and confirm the viability of the isolated PBMCs, and TSS was immediately performed.

#### **4. Procedures for generating various thermal assault conditions**

From the previously published study, 70 animals were placed into seven groups with 10 individuals each. In total, 70 Indian Zebu-Jersey crossbred cattle breed's blood samples yielded 70 aliquots of PBMCs, both stressed and unstressed cells. With the exception of the 0°C TAC, the PBMCs were exposed to each of the four TACs for 3 h and 6 h (0, 37, 40, and 45°C) (**Figure 2**). Before the TSS procedure, the number of viable cells were estimated to be between 7.04 106 -2.56 107 cells/mL. About 1 106 PBMCs/mL were present in each aliquot of 500 μL [1].

All PBMC aliquots were first stabilised for 30 min. at 37°C in a 5% CO2 incubator with nutritive medium (RPMI 1640; Cole-Parmer Binder C170UL-120V-R CO2 Incubator, Mumbai, India). After 30 min of initial stabilisation of both stressed and unstressed samples in nutrient media at 37°C in 5% CO2 incubator, the control sample labelled unstressed was immediately harvested.

#### **5. Procedures for thermal stress stimulation of PBMCs**

Isolated PBMCs were divided into two groups, one of which underwent TS and the other of which was not. Initially, aliquots of (500 μL) PBMCs were cultured in a nutrient medium (RPMI 1640) at 37°C for 30 min. in a 5% CO2 incubator for stabilization. Different TACs and DTEs were used to conduct an in vitro TSS of PBMCs. The PBMC aliquots (500 L) were labelled and put through four different TACs in a circulating REMI RSB-12 water bath, as illustrated in **Figure 3**, No TS: Control, 37°C: Normal temperature, 40°C: Moderate heat, and 45°C: Extreme heat) included of four treatment groups, whereas two DTEs were also included (3 and 6 h).

After TSS was completed, the stressed PBMCs were given time to recover at 37°C for 30 min in an incubator with 5% CO2 before being trypsinized and harvested. Unstressed control samples (500 μL) on the other hand, were stabilized for 30 min before being harvested. They were also not subjected to TAC or DET (0°C or 0 h). Both stressed and unstressed PBMCs will be used for total RNA isolation for heat shock protein gene expression analysis, including *HSP* 70 and 90 genes or other downstream analyses.

#### **6. Evaluation of the PBMC count and viability**

Using the Trypan blue dye exclusion method, PBMC number and viability were calculated after PBMC isolation. The trypan exclusion dye method involved the staining of the PBMCs with trypan blue dye such that the viable PBMCs were not stained but dead cells were stained and excluded when observed under a microscope on a haemocytometer (Microyn Improved Neubauer Haemocytometer, Hunt Valley,

**Figure 3.**

*Illustration of an experimental design with varied thermal assault conditions and durations thermal exposure for stressed and unstressed peripheral blood mononuclear cells of Indian Zebu-Jersey crossbred cattle [1].*

Maryland, USA) (CELESTRON Labs CB2000C Compound Microscope, Celestron, LLC., California, USA) (**Figure 2a**). Before TSS, it was estimated that there were 7.04 � 106 to 2.56 � 107 viable cells per millilitre. Total RNA was extracted from the isolated PBMCs. Using a Thermo-Scientific-Nano Drop 2000 spectrophotometer, total RNA was quantitated (Shimadzu co-operation, Kyoto, Japan).

#### **7. Estimation of viable peripheral blood mononuclear cells and quantitation of viable peripheral blood mononuclear cells**

As earlier published by Onasanya and his co-workers [1], the PBMCs were estimated, and viability was checked using various parameters shown in Eqs. (1)–(4).

%Viability of PBMCs ¼ Total viable PBMCs*=*Total number of PBMCs Viable ð Þ þ Dead �100 (1)

Average number of viable PBMCs per square

¼ Total number of viable PBMCs in 4 Squares*=*4 (2)

Dilution factor ¼ Total volume Volume of PBMCs þ Volume of trypan blue dye*=*Volume of PBMCs

(3)

Concentration of Viable PBMCs*=*Square ¼ Average number of viable PBMCs � Dilution factor � 104 (4)

For this study, two dilution factors were used.

In their previously published data (**Table 1**) on the TSS of PBMCs, Onasanya and his coworkers [1] found that heat shock/thermal assault at 45°C for 6 h-DTE had the greatest impact on PBMCs of Indian Zebu-Jersey crossbreds than at any other thermal conditions examined.

#### **8. Processing and preservation of Isolated PBMCs for the extraction of total RNAs and mRNA expression analyses**

PBMCs to meant for total RNA isolation, especially for gene expression analyses, should be aliquoted into convenient volumes, such as 250 μL of equal PBMCs per

*Means within the same column having different superscripts are significantly different \*\*\**P *< 0.001; TACs: Thermal Assault Conditions; DTEs: Durations of Thermal Exposures.*

#### **Table 1.**

*Mean values for PBMCs and RNAs after TSS at different TACs and DTEs in Indian-Jersey Crossbreds [1].*

*Techniques of Using Peripheral Blood Mononuclear Cells as the Cellular System… DOI: http://dx.doi.org/10.5772/intechopen.109431*

treatment group. The purpose of this is meant to eliminate error of orthogonality that could arise from variation in the numbers of PBMCs among treatment groups, so that variation in RNA concentration will not be due to differences in PBMC numbers among the treatment groups. Thereafter, the PBMC was centrifuged at 8000 rpm for 5 min, gently pipette out the supernatant without disturbing the pellet, add 250 μL of RNAlater® and store at 4o C for 24 h to stabilize the RNA and internal environment of the cells. After the cellular environment of the PBMC has stabilised for 24 hours, centrifuge the PBMCs at 10,000 rpm for 10 min to remove the RNAlater® gently without disturbing the PBMCs pellet and store the recovered PBMCs at �80°C for downstream analyses.

#### **9. Computation of equal number for PBMCs across the treatment groups**

How to guarantee that each treatment group has equal number of PBMCs is shown in **Table 2**. For instance, the maximum PBMC count in the four treatment groups is 3.08 �107 . Note that, the treatment group with the highest PBMC count will be used as the benchmark for the computation to guarantee that PBMC counts are equal across treatment groups. Eqs. (5–7) demonstrate the various equations to make the estimations.

$$\text{Number of PBMCs in 250 }\mu\text{L} = \frac{\text{PBMCs in 250 }\mu\text{L}}{250} \tag{5}$$

$$\text{P. Number PBMCs per 3.08} \times 10^7 = \frac{3.08 \times 10^7}{\text{PBMCs}/\mu\text{L}}\tag{6}$$

Volume (μL) to remove from PBMCs of each treatment to generate equal number PBMCs across the treatment groups

$$\mathbf{r} = 250 - \text{PBMCs per } 3.08 \times 10^7 \tag{7}$$

#### **10. Procedures for isolation of total RNA from PBMCs pellet**

**Table 2.**

*Computation of equal PBMC count across the treatment groups.*

subsequently cover the tube and mix thoroughly for 5 times in the right – left direction.

#### **11. How to preform TapeStation quantity control check for total RNA integrity for preparation of mRNA library and mRNA expression**

A 4150 TapeStation System (Catalog: G2992AA, Agilent) that is intended for analysing Eukaryote and Prokaryote RNA can be used to perform the RNA quality assessment [10]. Total RNA molecules with lengths between 50 and 6000 nt are used to compute the RNA integrity (RINe) values, which are used to assess the quality of the total RNA. 3μL of RNA ScreenTape were combined with 1 μL of total RNA sample. Sample buffer was heated at 72o C for 3 min to denature it, after which the sample was immediately put on ice for 2 min before being loaded onto the Agilent 4150 TapeStation equipment. The software assigns total RNA integrity number (RINe) that indicates the integrity of the total RNA [10]. RINe values were graded from 1 to 10, with values between 1 and 5 indicating fully degraded total RNA, 5–7 indicating moderately degraded total RNA, and values above 8 indicating high-quality total RNA. Total RNA whose RINe number falls within the range of 6 and above are of good quality hence they are recommended for preparation mRNA library and gene expression.

#### **12. Procedures for total RNA quantitation**

RNA concentration was determined on Qubit® 3.0 Fluorometer using the Qubit™ RNA BR Assay Kit (Catalog: Q10211, ThermoFisher Scientific), which contains RNA reagents consisting of buffers, dye that binds specifically to RNA with linear fluorescence detection in the range of 20 ng/ul to 1000 ng/ul and two RNA standards [11]. The dye and the buffer were diluted at 1:200 ratio and 1 μl of the RNA sample was mixed with the dye mix and incubated at RT for 2 min and the readings were taken in the Qubit.3 Fluorometer. Prior to the sample's measurement, the instrument was calibrated using the two standards provided in the kit [11] (**Table 3**).

#### **13. Conclusion**

The cellular systems of livestock animals are exposed to heat shock under prolonged and extreme TACs such high tropical temperatures, which prevents proper cell performance and may even cause cell death or prompt apoptosis. Consequently, severe TAC-DTE combinations have an adverse effect on cell count and survival by causing prompt apoptosis. Therefore, PBMCs can be employed as a cellular model or biological indicator to learn more about how animals' response to thermal assault conditions both *in vitro* and/or *in vivo*. In order to better understand how livestock animals, react to *in vitro,* it will be established in the future whether there is a relationship between the decreased PBMC count following *in vitro* TSS and the expression of the heat shock protein genes. This will enable better understanding of the thermotolerance ability of bovine species and other livestock animals under real-life scenarios/conditions for improved adaptability, survivability, and production performance, the biological data obtained from such study will be used to understand the *in vivo* response of livestock animals to different environmental TACs.

Finally, researches, academics, and livestock farmers will all profit greatly from the *in vitro* thermal stimulation and associated methodologies /procedures as presented in this chapter regarding the function PBMCs can play as a biological indicator in the monitoring and control of heat stress challenges in farm animals.

#### **Table 3.**

*TapeStation quality control check for total RNA quantitation and total RNA integrity values.*

### **Acknowledgements**

Some of the techniques discussed in this chapter was previously reported by Onasanya et al. (2022) and published Veterinary World. The full copy of the original article is available at www.veterinaryworld.org/Vol.15/September-2022/10.pdf We acknowledge TWAS-DBT Post-Doctoral Research fellowship program offered to Dr Gbolabo Olaitan Onasanya for supporting the laboratory techniques and research activities reported in this book-chapter.

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Gbolabo Olaitan Onasanya1,2,3\*, Aranganoor Kannan Thiruvenkadan2 , Alice Adishetu Yisa4 , Krishnaswamy Gopalan Tirumurugaan3 , Murali Nagarajan2 , Saravanan Ramasamy2 , Raja Angamuthu5 , George Mutani Msalya6 and Christian Obiora Ikeobi7

1 Department of Animal Science, Federal University Dutse, Dutse, Nigeria

2 Department of Animal Genetics and Breeding, Veterinary College and Research Institute, Tamil Nadu Veterinary and Animal Sciences University, Chennai, Tamil Nadu, India

3 Translational Research Platform for Veterinary Biologicals, Central University Laboratory, Tamil Nadu Veterinary and Animal Sciences University, Chennai, India

4 Department of Biochemistry, Federal University Dutse, Dutse, Nigeria

5 Department of Veterinary Microbiology, Veterinary College and Research Institute, Tamil Nadu Veterinary and Animal Sciences University, Chennai, Tamil Nadu, India

6 Department of Animal, Aquaculture and Range Sciences, Sokoine University of Agriculture, Morogoro, Tanzania

7 Federal University of Agriculture Abeokuta, Abeokuta, Nigeria

\*Address all correspondence to<EMAIL_ADDRESS>
© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

[1] Onasanya GO, Msalya GM, Thiruvenkadan AK, Murali N, Saravanan R, Raja A, et al. Exposure to high thermal conditions for a long time induces apoptosis and decreases total RNA concentration in peripheral blood mononuclear cells among Indian Zebu– Jersey crossbreds. Veterinary World. 2022;**15**(9):2192-2201

[2] Onasanya GO, Msalya GM, Thiruvenkadan AK, Sreekumar C, Tirumurugaan GK, Sanni TM, et al. Single nucleotide polymorphisms at heat shock protein 90 gene and their association with thermo-tolerance potential in selected indigenous Nigerian cattle. Tropical Animal Health Production. 2020;**52**(4):1961-1970

[3] Onasanya GO, Ikeobi CO, Ayotunde AS, Oke FO, Olorunisola RA, Decampos JS. Thermo-regulatory functions of heat shock protein genes in some selected tropically stressed livestock animals. International Journal of Applied Research and Technology. 2017;**6**(8):37-43

[4] Habeeb AAM, Gad AE, Tarabany AA, MAA A. Negative effects of heat stress on growth and milk production of farm animals. Journal of Animal Husbandry Dairy Science. 2018;**2**(1):1-12

[5] Kishore V, Sodhi M, Sharma A, Shandilya UK, Mohanty A, Verma P, et al. Transcriptional stability of heat shock protein genes and cell proliferation rate provides evidence of superior cellular tolerance of Sahiwal (*Bos indicus*) cow PBMCs to summer stress. Review Journal of Veterinary Science. 2016;**2**(1):34-40

[6] Siddiqui SH, Subramaniyan SA, Kang D, Park J, Khan M, Choi HW, et al. Direct exposure to mild heat stress

stimulates cell viability and heat shock protein expression in primary cultured broiler fibroblasts. Cell Stress Chaperones. 2020;**25**(6):1033-1043

[7] Gao C, Zhao Y, Li H, Sui W, Yan H, Wang X. Heat stress inhibits proliferation, promotes growth, and induces apoptosis in cultured Lantang swine skeletal muscle satellite cells. Journal of Zhejiang University Science B. 2015;**16**(6):549-559

[8] Wang J, Wei Y, Zhao S, Zhou Y, He W, Deng W, et al. The analysis of viability for mammalian cells treated at different temperatures and its application in cell shipment. PLoS One. 2017;**12**(4):e0176120

[9] Siddiqui SH, Subramaniyan SA, Kang D, Park J, Khan M, Shim K. Modulatory effect of heat stress on viability of primary cultured chicken satellite cells and expression of heat shock proteins *ex vivo*. Animal Biotechnology. 2021;**32**(6):774-785

[10] Agilent Technologies, Inc. Agilent Technologies Hewlett-Packard-Straße 8 76337. Waldbronn, Germany: Agilent Technologies; 2015. pp. 2013-2014

[11] ThermoFisher Scientific, Waltham, Massachusetts, U.S.A. https://www.the rmofisher.com/document-connect/doc umentconnect.html?url=https%3A%2F %2Fassets.thermofisher.com%2FTFS-Assets%2FLSG%2Fmanuals%2Fmp114 90.pdf&title=UXVhbnQtaVQgUmlib0d yZWVuIFJOQSBSZWFnZW50IGFuZC BLaXQ=

#### **Chapter 5**

## Factors Characterizing Puberty in Ram Lambs of Four Breeds Raised under High Altitude Conditions

*Harvey Lozano, Jimmy Vargas, Liliana Chacón and Nathalie Kirschvink*

#### **Abstract**

The aim of this study was to compare ram lambs of four Colombian wool breeds raised under high altitude conditions to describe evolution of semen characteristics, body development, and libido and plasma testosterone. Corriedale, Hampshire, Romney Marsh, and Creole rams were enrolled since the age of 4 months for libido and testosterone (maximum, mean and amplitude) assessment, whereas semen collection was performed between 6 and 11 months of age by use of electro-ejaculation. Beside analysis of variables in function of breed and over time, a semen maturity score, considering semen volume, mass motility, individual progressive motility, concentration and % of living spermatozoa was established in function of adult rams' reference data. Colombian Creole displayed significantly higher results regarding all variables and showed the most important body development at each time point of the study.

**Keywords:** male lamb, non-seasonal, puberty, maturity, semen, libido, testosterone, breed

#### **1. Introduction**

Puberty corresponds to the transition into adulthood and can be defined in rams as the time when fertile spermatozoa are present in the ejaculate [1]. For some authors it is defined as the time when rams show 'interest' in females in estrus by successive mounting with ejaculation [2].  Another definition of puberty in ram lambs is based on a sustained rise in plasma testosterone concentrations over three consecutive blood samplings performed in one week and confirmed by the presence of spermatozoa in the ejaculate with at least 30% mass motility [1, 4]. In general, sexual development of ram lambs appears to be more closely associated with body growth than with chronological age. Body weight can be a good criterion for the achievement of puberty than chronological age alone [2]. Rate of testis growth is reported to be more rapid in lambs of highly prolific breeds such as the Finnish Landrace than in less prolific breed [5]. Louda et al, after observing testis growth and small but consistent differences in the development of sexual activity and of sperm production, suggested that young rams of prolific breeds (Romanov and Finnish Landrace) may differ in their future reproductive performance. The early pubertal development associated with increased body weight is desirable in terms of improved reproductive performance [5, 6]. Broad information about the onset of puberty is of considerable importance for successful reproductive management [7]. On one hand, it is important to avoid inbreeding by separating prepubertal rams from ewes, especially in counties with extensive breeding practices and poor separation between animal groups of different ages or different reproduction status. On the other hand, insufficient sexual maturity of young rams may lead to reduced flock fertility. A few information is available in terms of testosterone levels, semen quality, libido and sexual performance at high altitude conditions in a non-seasonal country [8].

Seasonality is a main effect in the onset of rams' puberty. Animals live under the influence of seasonal fluctuations of environmental conditions with variable amplitudes frequently more marked in the higher latitudes and altitudes [9, 10]. Photoperiod is the key environmental signal timing in the reproductive cycle because the effect of season and/or day length has been studied as a main factor in onset of puberty in different breeds of young rams. Rams'sensitivity to photoperiod is different from ewes. Reproductive season can be influenced by birth date and puberty time. During spring and summer, a persistent hyper prolactinaemia was associated with low circulating FSH concentrations in Texel rams [11]. Young rams may reach puberty by 4 months of age during the first fall season, while the onset of puberty may be delayed until 9–12 months for rams born late in the lambing season and if additionally, the situation is accompanied of poor feeding and poor climatic conditions [12, 13]. In Karakul rams in southern Iran is observed how lambs born at the beginning of spring is sexually more precocious than lambs born later [14].

It is well known that interactions between body and testis growth, sperm production and testosterone secretion is complex since early pre-pubertal ages and, in many aspects, such events can be influenced by both the genetic background of animals and the environment where they are raised [2]. Plasma FSH concentrations were lower in Texel rams than in Suffolk and Ile-de-France rams during both pre-pubertal and pubertal periods [11]. Emsen [5] showed that crossing Awassi sheep with a native breed Redkaraman with relatively higher growth rate can considerably improve the early pubertal development of rams. Puberty of Awassi ram lambs in improved flocks, started around seven months of age and at on average weight of 34.6 kg [15]. In general, sexual development of ram lamb appears to be more closely associated with body growth than with chronological age and the live weight of the ram lambs at puberty is probably related to the genotype [2]. In that research ram lambs of breeds Friesland and Chios attained puberty when they reached approximately 50% of their adult body weight. By comparing different breeds and hormone levels during pre and pubertal time it was shown that Canadian ram lambs had significantly lower FSH levels than other breeds like Finnish Landrace. However, as adults the same group of Canadian rams had larger testicles and better semen quality [12]. In pubertal development, scrotal circumference was highly and positively correlated with live weight, but negatively correlated with inhibin and FSH concentrations in rams of Suffolk and DLS (Dorset Leicester Suffolk) [13]. Rate of testis growth was more rapid in lambs of Finnish Landrace with high prolificacy than a less prolific breed such as Merino [16].

Onset of puberty time in ram lambs is considerably influenced by nutrition. Prepubertal nutritional restrictions delay testicular growth and the rate of sexual development in ram lambs is highly dependent on energy intake and live weight gain [17].

*Factors Characterizing Puberty in Ram Lambs of Four Breeds Raised under High Altitude… DOI: http://dx.doi.org/10.5772/intechopen.110264*

Nutritional restrictions can influence the activity of the hypothalamus-pituitary axis and thus reduce gonadotropin levels in ram lambs. It can even be influenced during prenatal period based on mother's nutrition during pregnancy reducing lamb's pituitary capacity to liberate gonadotropins like LH [17]. The scrotal circumference of rams under an intensive feeding management increased by 4.4%, while decreased for rams under extensive conditions by 2% due to poor nutritional conditions [18].

The social environment in which male lambs are reared appears to influence some aspects of reproductive behavior: the exposure to cyclical ewes during the first 6 months of life is fundamental to induce an increase in testosterone concentrations and in testicular size and also the social rank of male lambs during pre-pubertal development affected reproductive performance of adult rams [19].

Sexual performance of rams having previously direct contact with females at 7– 9 months of age was enhanced in comparison with rams without that previous experience [20]. In a study developed to evaluate the possible influence of litter size on the onset of puberty and hormone levels it could be realized how lambs born as singletons had lower testosterone levels at 8 months of age than those born as twin or triplets [12]. Ungerfeld and González-Pensado [21] reported a study in which intensive malemale sexual behavior is described in pubertal lambs with approximately the same age (less than 6 months) and it was shown how more dominant lambs displayed more intensive sexual behavior toward subordinate males and cyclical ewes, whereas more subordinate males received more mounts from other males and were less active to mount. Ram lambs of the breed Polish Milk Sheep, which is more prolific and attains puberty earlier, were more active in sexual play than ram lambs of the Polish Whiteheaded Mutton [22].

#### **2. Materials and methods**

#### **2.1 Animals and location**

The study was conducted between July 2012 and April 2013 at the experimental farm of the National University of Colombia at 2650 m of altitude at 4°420 latitude north and 74°120 longitude west, near Bogotá (Colombia).

Twenty-four ram lambs, aged 3–4 months at the beginning of the trial and from four different breeds (6 per breed) were enrolled in the study. Animals belonged to the native Colombian Creole and three foreign adapted breeds that are frequently used by Colombian breeders: Hampshire, Corriedale and Romney Marsh. The animals were born in the center and were selected of base of a selection index. They were grazing all the time of the experiment and each animal received daily 200 g of a pelleted concentrate mixture and 300 g of trefoil hay. Mineral salts and water were provided ad libitum.

The protocol was approved by the Bioethics committee of the Faculty of Veterinary Medicine and Animal Science of the National University of Colombia (Acta: CD-071-2014).

#### **2.2 Assessment of semen characteristics in ram lambs**

Once per month, ram lambs were collected by electro ejaculation, penis was exposed from the prepuce cavity and the urethral process was gently introduced into a conical tube previously to start ejaculation process. Semen was placed in a water bath

at 37°C and subjected to the following tests: (1) Volume was measured in a conical glass tube graduated with 0.1 mL optically visible intervals; (2) motility (Mass: semen was assessed for semen wave motion graded on a subjective scale ranging from 1 to 5, where 1 was scored when there was no mass movement and 5 represented vigorous waves of sperm motion and Individual progressive motility %).

Semen concentration was determined using a standard spectrophotometer (540 nm). (4) The proportion of live and dead spermatozoa was determined using the nigrosine-eosin staining technique by counting at least 200 spermatozoa under oil immersion objective (1000) random fields. The proportion of morphologically abnormal spermatozoa was also determined by examining 200 spermatozoa in an eosin-nigrosine smear under the same magnification. Abnormal spermatozoa were then classified into proportion of spermatozoa with head abnormalities, midpiece abnormalities, tail abnormalities, proximal droplet, distal droplet, detached heads or tailless spermatozoa [23]. All examinations were performed by the same operator.

#### **2.3 Plasma testosterone concentration**

Blood samples were collected every month during 5 hours at 30 minutes intervals using heparinized venoject® tubes and centrifuged (1500 g for 15 min). The plasma was recovered and stored at 70°C. The concentration of testosterone in the plasma samples was measured in duplicate by an adapted enzyme immunoassay using a diagnostic commercial kit (DS-EIASteroid®-Testosterone RH-353). A calibration curve was used in order assess concentrations ranging from 1.25 to 40 nmol/l of testosterone in plasma. Known samples of a mature adult ram and of an ovariectomized ewe were used as positive and negative control respectively on each plate. Lower detection limit was 0.2 nmol/l and inter-and intra-assay were 6.7 and 5.5%, respectively. In each ram, testosterone curves were plotted over each 5 h period to determine the maximum value. The difference between recorded maximum and minimum concentration allowed to calculate testosterone amplitude.

#### **2.4 Body measurements**

Body weight was assessed once monthly using an electronic balance. Scrotal circumferences using a scrotal tape was evaluated also monthly.

#### **3. Statistical analysis**

Semen characteristics, testosterone concentration (peaks, amplitude, mean) were analyzed using a generalized linear model (GLM) for repeated measures analysis. The model contained effects due to breed and period. Mean differences in body weight (kg), scrotal circumference (cm) and scrotal circumference/body weight was compared by Duncan's method. The level of significance was set at P < 0.05 for all tests. Data was analyzed using SAS System (SAS version 9.12, SAS Institute Inc.). A semen maturity score, aiming at comparing the global semen quality of young rams in function of semen quality of adult rams of the same breed, was established as follows: Semen characteristics of five adult rams of each breed collected over a period of one year was averaged and considered as reference value. These breed-specific reference values were established for semen volume, concentration, mass motility (MM), individual progressive motility (IPM) and percentage of living spermatozoa. At each

*Factors Characterizing Puberty in Ram Lambs of Four Breeds Raised under High Altitude… DOI: http://dx.doi.org/10.5772/intechopen.110264*

month of collection, semen variables of the ram lambs were expressed as % in function of the adult rams' reference value. This percentage was classified in function of three semen maturity levels: when 50, 75 or 100% of the adults' value were reached, a note of 1 was accorded to this semen variable. If the % was lower than 50, 75 or 100%, a note of 0 was attributed. At each month of sampling, the maturity score establishing the sum of the notes attributed for semen volume, concentration, MM, IPM and % of living spermatozoa for achievement for 50, 75 or 100% of adult rams'semen quality was calculated.

#### **4. Results**

**Table 1** shows semen characteristics and body measurements in function of breed and indicates significant time-, breed- or interaction effects. No breed effect was found for semen volume, whereas Corriedale rams showed lowest values for concentration, MM, IPM ad % of normal spermatozoa (p < 0.05). **Figure 1** displays detailed evolution of these variables (volume, MM, IPM, concentration and % of normal spermatozoa) over time and in function of breed. All variables achieved a stable level when the rams were 10 and 11 months old. Creole rams showed consistently higher values for all variables except semen volume, whereas Corriedale rams generally showed the lowest values.

**Table 2** shows the evolution of the semen maturity score per breed and over time. Although no significant differences between breeds were found, a clear trend for more rapid development were found in Creole and Romney Marsh ram lambs.

**Table 3** shows as the evolution of body development (expressed in function of adults' ram weight) over time. Creole and Romney Marsh display highest values at begin of the investigation whereas Creole rams maintained this rapid development until the age of 11 months.

**Figure 2a** and **b** display maximum testosterone concentration and testosterone amplitude in function of breed and over time. Creole ram lambs had significantly higher values. The timerelated increase was mostly observable in all breeds between 3 and 6 months of age. There were not correlations between testosterone levels and semen quality, sexual activity, or body measurements. In general, the first expressed signs of interest in the oestrous females occurred prior to the sustained rise in testosterone.

#### **5. Discussion**

This study aimed at describing semen characteristics, body development, and libido and plasma testosterone concentration in young rams of four economically important wool breeds in high altitude conditions in Colombia. Prior considering and discussing results in detail some drawbacks must be mentioned. Semen collection was performed by electro-ejaculation since the age of 6 months and as expected and described by others [1, 18], semen quality and testosterone levels recorded at this age suggested that puberty was already achieved by most of the rams. The relatively late start of semen collection was due to the fact that despite regular training of the rams, semen collection by use of an ewe in estrus was not successful in all rams, which meant that semen quality data were only available in a few rams. Therefore, the investigators decided to switch to semen collection by use of electro-ejaculation. Even

 **1.** *Mean ( SD) seminal characteristics and body measure evaluated in ram lambs in four breed (Creole, Romney, Corriedale and Hampshire).* *Factors Characterizing Puberty in Ram Lambs of Four Breeds Raised under High Altitude… DOI: http://dx.doi.org/10.5772/intechopen.110264*

#### **Figure 1.**

*Evolution of semen quality in rams of four breeds (7 animals/breed) over time in rams aged between 6 and 11 months. A significant time effect was found for semen volume (a), whereas significant time and breed effects were found for mass motility (b), individual progressive motility (c), concentration (d) and % of living spermatozoa (e). Data are shown as mean and standard deviation.*

#### **Table 2.**

*Semen maturity scores of Corriedale, Creole, Hampshire and Romney Marsh ram lambs tested between 6 and 11 months of age.* *Factors Characterizing Puberty in Ram Lambs of Four Breeds Raised under High Altitude… DOI: http://dx.doi.org/10.5772/intechopen.110264*

*Data are expressed as % in functions of adult rams' body weight of the same breed. Means and SD are shown for each breed.*

*CO, Corriedale; CR, Creole; HA, Hampshire; RM, Romney Marsh.*

#### **Table 3.**

*Evolution of body development in function of breed and over time.*

if the semen characteristics do not importantly change when natural versus electric ejaculation are compared this methodological difference must be kept in mind when ram lamb semen data are expressed in function of adults'semen data, leading to an underestimation of young rams'semen quality. Although the semen quality score allowed to consider simultaneously all important semen characteristics (volume, MM, IPM, concentration and % of living spermatozoa) and thereby allowed to assess the ability of young rams at the end of the puberty (rather than to consider each variable separately), it remains a matter of fact that onset of puberty occurred before or just around first semen collection and that a precise age of onset of puberty could not be established for each ram and/or each breed. Regarding the end of the study, it appears that albeit a stabilisation since at least two months of all parameters (except semen concentration), it is impossible to define time point sexual maturity. As evidenced by the semen quality score, only two Creole rams showed semen characteristics that perfectly corresponded to those of adults of the same breed. The study should have been prolonged to assess when semen maturity was reached. Nevertheless, the present study describes that Colombian Creole appears as the most precocious breed: indeed, semen variables, semen maturity score, behavioural aspects as well as plasma testosterone levels were significantly increased in comparison to the other breeds, especially to Corriedale rams whose performances were almost always lower than in the other breeds. At the same time, it became apparent that development was more important in Creole rams whose relative body weight equalled 55–56% at the age of 6 months (versus 47–51% in the other breeds) and 80–85% at the age of 11 months (versus 55– 65% in the other breeds). Moreover, when considering semen quality data (**Figure 1**), it can be said that since the age of 8 months almost all rams, except some Corriedale rams whose semen concentration remained below 2000 million spermatozoa/ml, were able to ensure reproduction. Indeed, the only limiting factor in terms of reproductive capacity seemed to be concentration.

It is interesting to state that this local breed appears to be optimally adapted to high altitude Andes conditions, whereas the foreign breeds, although imported since more than 50 years, show significant differences. They mostly account for a larger body size, meaning that the minimal % of body development is achieved later point, but seem also to depend on other factors. Indeed, testosterone levels were significantly higher in Creole rams and might impact semen quality. Another aspect was evaluated,

#### **Figure 2.**

*(a) Maximum plasma testosterone concentrations assessed between the age of 3 (July data) and 10 (March data) months in Creole, Romney Marsh, Hampshire, and Corriedale ram lambs. Data are shown as means with SD. A significant collection effect was recorded over time, as well a significant breed effect with Creole rams showing significantly higher testosterone levels, p < 0.05. (b) Plasma testosterone amplitude assessed between the age of 3 (July data) and 10 (March data) months in Creole, Romney Marsh, Hampshire, and Corriedale ram lambs. Data are shown as means with SD. A significant collection effect was recorded over time, as well a significant breed effect with Creole rams showing significantly higher testosterone levels, p < 0.05.*

but results are not presented here, it is about sexual behavior of the ram lambs since they were three until one year of age.

In Colombia some time after this research was developed, this group could perform a trial under low altitude conditions and ram lambs belonged to hair breeds instead of wool, finding some results in concordance to these results, especially about testosterone levels during ram lambs growing up. Into that hair groups experiment, time of evaluation was shorter than this research, but some parameters could be compared and the new information was useful for the good understanding of puberty *Factors Characterizing Puberty in Ram Lambs of Four Breeds Raised under High Altitude… DOI: http://dx.doi.org/10.5772/intechopen.110264*

in ram lambs in a country as Colombia where there is different altitude in farms despite of being a non-seasonal country [24].

In conclusion, this investigation describes how semen quality, libido and plasma testosterone evolve over time an in four Colombian wool breeds and allows to point out the importance of body development to achieve satisfying reproductive abilities. It was shown that almost all variables were improved in Colombian Creoles, whereas Corriedale showed the lowest development.

#### **Author details**

Harvey Lozano1 \*, Jimmy Vargas2 , Liliana Chacón3 and Nathalie Kirschvink4

1 Universidad Nacional de Colombia, Facultad de Medicina Veterinaria y de Zootecnia, Sede Bogotá, Bogotá, Colombia

2 Instituto de Genética, Universidad Nacional de Colombia, Bogotá, Colombia

3 Facultad de Ciencias Agropecuarias, Universidad de La Salle, Bogotá, Colombia

4 Faculty of Medicine, Namur Research Institute for Life Sciences (NARILIS), University of Namur, Namur, Belgium

\*Address all correspondence to<EMAIL_ADDRESS>
© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

[1] Kridli RT et al. Sexual activity and puberty in pure Awassi and crosses and backcrosses with Charollais and Romanov sheep breeds. New Zealand Journal of Agricultural Research. 2007; **50**(4):429-436

[2] Belibasaki S, Kouimtzis S. Sexual activity and body and testis growth in prepubertal ram lambs of Friesland, Chios, Karagouniki and Serres dairy sheep in Greece. Small Ruminant Research. 2000;**37**(1–2):109-113

[3] Price EO, Estep DQ, Wallach SJ, Dally MR. Sexual performance of rams as determined by maturation and sexual experience. Journal of Animal Science. 1991;**69**(3):1047-1052

[4] Wood RI et al.  Biology of Reproduction. 1991;**45**(1):82-88

[5] Emsen E. Testicular development and body weight gain from birth to 1 year of age of Awassi and Redkaraman sheep and their reciprocal crosses. Small Ruminant Research. 2005;**59**(1):79-82

[6] Elmaz Ö, Dikmen S, Cirit Ü, Demir H. Prediction of postpubertal reproductive potential according to prepubertal body weight, testicular size, and testosterone concentration using multiple regression analysis in Kıvırcık ram lambs. Turkish Journal of Veterinary and Animal Sciences. 2008;**32**(5):335-343

[7] Kridli RT, Al-Yacoub AN. Sexual performance of Awassi ram lambs reared in different sex composition groups. Applied Animal Behaviour Science. 2006;**96**(3–4):261-267

[8] Avellaneda Y, Rodriguez F, Grajales H, Martinez R, Vasquez R. Puberty determination in rams according to body characteristics, and evaluation of quality of ejaculate and testosterone. Livestock Research for Rural Development. 2006; **18**(10):61-67

[9] Valencia J, Trujillo MJ, Espinosa MA, Arroyo J, Berruecos M. Pubertad en corderos Puberty in Pelibuey lambs born of sheep with seasonal or continuous reproduction. Revista Científica de Maracaibo. 2005;**15**(005):432-438

[10] Rosa HJD, Bryant MJ. Seasonality of reproduction in sheep. Small Ruminant Research. 2003;**48**(3):155-171

[11] Mandiki SNM, Derycke G, Bister JL, Paquay R. Influence of season and age on sexual maturation parameters of Texel, Suffolk and Ile-de-France rams: 1. Testicular size, semen quality and reproductive capacity. Small Ruminant Research. 1998;**28**(1):67-79

[12] Langford GA, Ainsworth L, Marcus GJ, Shrestha JN. Photoperiod entrainment of testosterone, luteinizing hormone, follicle-stimulating hormone, and prolactin cycles in rams in relation to testis size and semen quality. Biology of Reproduction. 1987;**37**(2):489-499

[13] Sanford LM, Moore C, Voglmayr JK, Fahmy MH. Sexual maturational changes in circulatory inhibin concentration in relation to FSH concentration and testicular size in Suffolk and DLS rams. Theriogenology. 2000;**54**(5):719-730

[14] Kafi M, Safdarian M, Hashemi M. Seasonal variation in semen characteristics, scrotal circumference and libido of Persian Karakul rams. Small Ruminant Research. 2004;**53**(1–2):133-139

[15] Salhab SA, Zarkawi M, Wardeh MF, Al-Masri MR, Kassem R. Development

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of testicular dimensions and size, and their relationship to age, body weight and parental size in growing Awassi ram lambs. Small Ruminant Research. 2001; **40**(2):187-191

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[17] Bielli A, Katz H, Pedrana G, Gastel MT, Moraña A, Castrillejo A, et al. Nutritional management during fetal and postnatal life, and the influence on testicular stereology and Sertoli cell numbers in Corriedale ram lambs. Small Ruminant Research. 2001;**40**(1):63-71

[18] Fourie PJ et al. Scrotal, testicular and semen characteristics of young Dorper rams managed under intensive and extensive conditions. Small Ruminant Research. 2004;**54**(1–2):53-59

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[24] Chacón L, Lozano H, Orozco J, Ardila A. Characteristics of the puberty in hair ram lambs and its crosses in Colombia under low altitude conditions. Journal MVZ Cordoba. 2019;**24**(1):7097- 7103

### *Edited by Xiaojun Liu and Hong Li*

Healthy and sustainable animal husbandry is the goal of animal breeding. This book provides a detailed overview of applications of post-genome era techniques for livestock genetics and breeding. It also describes techniques for genetically selecting highly productive animals without producing large amounts of greenhouse gas emissions, community-based breeding programs for breeding rams and bucks, the response of bovine species to in vitro thermal stress stimulation, and the semen characteristics of wool-breed ram lamb-raised in conditions of high altitude. The book not only presents advanced animal genetic and breeding methods but also highlights useful, practical technology for animal breeding under specific climate, geographic, and environmental conditions.

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Published in London, UK © 2024 IntechOpen © Nemika\_Polted / iStock

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*Edited by Xiaojun Liu and Hong Li*


Fronto-temporal lobar degeneration (FTLD) is a neurodegenerative disorder characterized by behavioral abnormalities, language impairment, and deficits in executive functions as the most typical clinical features (Seelaar et al., 2011). FTLD is clinically heterogeneous, as different clinical variants have been carefully described. On the basis of presenting clinical symptoms, behavioural variant FTD (bvFTD), agrammatic variant of Primary Progressive Aphasia (avPPA), and semantic variant of PPA (svPPA) represent the most common phenotypes (Benussi et al., 2015). Each one presents specific neuroimaging hallmarks; bvFTD is characterized by mesial and dorsolateral frontal damage, prevalent on the right side, avPPA is defined by involvement of Broca's area and left insula, whilst svPPA usually presents left rostral temporal involvement (Lashley et al., 2015).

Moreover, FTD is chacterized also by early stages, usually named mild cognitive impairment (MCI), that are still not completely characterized. Unfortunately, the diagnosis of FTD can be

difficult because of its insidious and gradual onset and can also be misdiagnosed as Alzheimer's disease (AD) (Rankin et al., 2005; Walker et al., 2005).

The discovery of mutations in GRN as a genetic determinant for FTLD resulted in the rapid identification of a large number of families carrying GRN mutations, inherited in an autosomal dominant pattern. At present, 77 different mutations in more than 240 unrelated families have been described (Ghidoni et al., 2012a). Mutations in GRN have been associated with a broad spectrum of clinical phenotypic variability (Benussi et al., 2008; Rohrer et al., 2010a,b).

GRN null mutations cause protein haploinsufficiency, leading to a significant decrease in progranulin (PGRN) levels that can be detected in plasma, serum, and cerebrospinal fluid of mutation carriers (Ghidoni et al., 2008, 2012b; Finch et al., 2009; Sleegers et al., 2009). It has been reported that PGRN promotes neuronal survival and neurite outgrowth in cultured neurons (Van Damme et al., 2008) and enhances neuronal survival under stress conditions (Kessenbrock et al., 2008). Data from GRN knockout experimental models suggest that PGRN deficiency leads to reduced synaptic connectivity and impaired plasticity, which may be contributing factors to FTLD pathology in human patients (Tapia et al., 2011; Petkau et al., 2012). Brain activity could be widely and variously affected in FTLD patients with GRN mutations. Neuroimaging studies have shown that the topography of brain atrophy is frequently asymmetric, and predominantly involves the frontal, temporal, and parietal cortex (Whitwell et al., 2012). Nevertheless, a recent study (Caroppo et al., 2014) have demonstrated that a diffuse and bilateral white matter involvement is common in patients with GRN mutations. The presence of white matter lesions is not surprising since expression of PGRN, not only in neurons but also in activated microglia, in astrocytes and oligodendroglia, has been previously ascertained (Ahmed et al., 2010). As a consequence, cortical and subcortical loop are both implicated in the disruption of intrinsic brain networks in GRN mutations carriers.

Electroencephalography is a reliable and non-invasive tool for the study of brain networks in dementia.  In particular, alpha and theta field potentials are deeply involved in tuning the large and local scale networks interactions in cognitive and psychiatric illnesses (Koenig et al., 2001; Tenke et al., 2011). Previous EEG studies have demonstrated peculiar modifications in brain oscillations in patients with MCI due to AD as compared to non-AD converters (Moretti et al., 2011a). Moreover, these changes in brain oscillations have been correlated with temporo-parietal and hippocampal atrophy (Moretti et al., 2007a, 2008a, 2009a,b, 2011a, 2012) as well as to white matter lesions (Moretti et al., 2007b, 2008b). In the present explorative study, we test the working hypothesis that changes in EEG oscillations could specifically detect different stages of FTLD, namely MCI-FTD versus overt FTD, as well as differences between GRN mutation carriers versus non- carriers. The search for new biomarkers is of great importance for an early diagnosis and for monitoring the effectiveness of new therapies.

#### MATERIALS AND METHODS

#### Subjects

#### Diagnostic Criteria

Clinical diagnosis of FTLD and LBD were made according to international guidelines (McKeith et al., 1996; Neary et al., 1998) as well as more recent revised criteria for the diagnosis of frontotemporal dementia (Gorno-Tempini et al., 2011; Rascovsky et al., 2011). MCI-FTD patients were recruited with mini mental state examination (MMSE) score higher than 24/30. Eleven patients with MCI-FTD, eight FTLD patients carrying GRN mutations, and 20 FTLD patients not carrying GRN mutations were included in the present study (**Table 1**). The diagnosis of the enrolled subjects is reported in **Table 2**. All patients underwent a series of standardized diagnostic and severity instruments, including: the MMSE (Folstein et al., 1975), the Clinical Dementia Rating Scale (CDRS; Hughes et al., 1982), the Hachinski Ischemic Scale (HIS; Rosen et al., 1980), the Instrumental and Basic Activities of Daily Living (IADL, BADL; Lawton and Brody, 1969) and a comprehensive neuropsychological battery (Lezak et al., 2004). The patients were recruited only with apparently primary cognitive deficits excluding psychic comorbid conditions like anxiety, depression, psychosis etc., or physical comorbidities like hypothiroidism, uncontrolled diabetes, vitamin B12, and folate deficiency, uncontrolled heart disease or hypertension, drug addiction, or alcohol abuse. Moreover, none of the patients was taking any drugs that might affect the EEG, namely psychoactive drugs, including acetylcholinesterase inhibitors or other drugs enhancing brain cognitive functions. In addition, patients underwent diagnostic neuroimaging procedures (magnetic resonance imaging, MRI), and laboratory testing to rule out other causes of cognitive impairment. In particular, MRI was able to exclude patients with major cerebrovascular diseases or other diseases (like tumors) that might influence EEG frequency rhythms. On the whole, 13 patients with primary progressive non-fluent aphasia (PNFA), 12 patients with the behavioral variant of FTD (FTD-bv), one patient with the semantic variant of FTD (FTD-sv), and one patient with Lewy Body Disease (LBD) were recruited. Demographic and clinical features of the sample in study are reported in **Table 2**. All experimental protocols had been approved by the local ethics committee of Scientific Institute for Research and Care (IRCCS) of Alzheimer's and psychiatric diseases 'Fatebenefratelli' in Brescia, Italy (Protocol numbers: 26/2014; 50/2015). Written informed consent was obtained from all participants or their caregivers, according to the Code of Ethics of the World Medical Association (Declaration of Helsinki).

#### EEG Recordings

The EEG activity was recorded continuously from 19 sites by using electrodes set in an elastic cap (Electro-Cap International,

Moretti et al. EEG and FTD

TABLE 1 | ANOVA results of demographic variable (age, education) MMSE score, and of PGRN dosage, high alpha, and theta EEG power of the study sample.

TABLE 2 | Clinical diagnosis of the patients recuited in the study.

FTD, fronto-temporale dementia; PPA, primary progressive aphasia; bv, behavioral variant; LBD, Lewy body dementia

Inc. Eaton, OH, USA) and positioned according to the 10– 20 international system (Fp1, Fp2, F7, F3, Fz, F4, F8, T3, C3, Cz, C4, T4, T5, P3, Pz, P4, T6, O1, and O2). All recordings were obtained in the morning with subjects resting comfortably. The patients were instructed to sit with closed eyes and relax and vigilance was continuously monitored in order to avoid drowsiness by an operator.  The left and right mastoids served as the reference points for all electrodes. The recordings were used off-line to re-reference the scalp recordings to the common average. Rereferencing was done prior to the EEG artifact detection and analysis. Data were recorded with a band-pass filter of 0.3– 70 Hz and digitized at a sampling rate of 250 Hz (BrainAmp, BrainProducts, Munich, Germany). Electrode-skin impedance was set below 5 k. Horizontal and vertical eye movements were detected by electrooculogram (EOG). The recording lasted 5 min with the subjects' eyes closed. EEG data were then analyzed and fragmented off-line in consecutive epochs of 2 s with a frequency resolution of 0.5 Hz. The average number of epochs analyzed was 140, ranging from 130 to 150. The epochs with ocular, muscular, and other types of artifacts were discarded by two skilled electroencephalographists. The spectral power we obtained is an estimation of a spectrum collapsed all over the scalp electrodes. In this way, the eventual contribution of the muscular or other artifacts is strongly reduced. Moreover, two skilled electroencephalographists checked separately the EEG traces (Moretti et al., 2003, 2004).

#### Analysis of Frequency Bands

A digital FFT-based power spectrum analysis (Welch technique, Hanning windowing function, no phase shift) computed the power density of EEG rhythms (ranging from 2 to 45 Hz) with a 0.5 Hz frequency resolution. The frequency bands range was computed on fixed limit as follows: (1) delta, 1–3 Hz; (2) theta, 4–7 Hz; (3) alpha 1 ore low alpha, 8–10,5 Hz; (4) alpha 2 ore high alpha, 10.5–14 Hz. These frequencies were computed on the power spectra averaged across all recording electrodes.

#### Genetic Analysis

GRN gene was analyzed in patients with FTLD and LBD by direct sequencing of all exonic and flanking intronic regions as previously described (Benussi et al., 2008). Plasma PGRN levels were measured in duplicate using an ELISA kit (Human PGRN ELISA Kit, AdipoGen Inc., Seoul, Korea) as previously reported (Ghidoni et al., 2012b). GRN positive carriers belongs to different families. This choice has been made to avoid that EEG patterns could be due to familial relationships. **Tables 2** and **3** shows the clinical diagnosis and related genetic mutations of GRN positive carriers.

#### Statistical Analysis

One-way analysis of variance (ANOVA) has been performed to compute the differences between groups (independent variables) in sociodemographic, MMSE score, PGRN dosage, and EEG oscillations (dependent variables) and neuropsychological tests. Age, education, and MMSE score have been used as covariates in ANOVA of brain rhythms to avoid confounding factors.

#### RESULTS

**Table 1** shows ANOVA significant results. As expected, the PGRN dosage was significantly different, with an impressive decrease in GRN null mutation carrier patients [F(19,77), df = 1, p < 0.001], confirming previous results (Ghidoni et al., 2012b). As about the EEG rhythms, results show: 1) a significant increase of high alpha power in GRN positive mutations carriers [F(4,22), df = 2, p = 0.003]; (2) a significant increase of theta power in GRN negative patients [F(3,14), df = 2, p = 0.002).

ANOVA analysis showed a decrease of the theta and alpha spectral power in MCI-FTD as compared to both PGRN positive

TABLE 3 | Genetic mutations identified in the GRN mutations carriers group and related clinical diagnosis.

FTDsv, fronto-temporal dementia semantic variant; FTDbv, fronto-temporal dementia behavioral variant; FTD-PPA, fronto-temporal dementia primary progressive aphasia; LBD, Lewy, body disease.

Moretti et al. EEG and FTD

and negative FTD patients. Given that the difference between PGRN positive and negative FTD patients were limited to theta and alpha power spectra, other frequency bands were not considered in the analysis.

The only significant results about neuropsychological battery were obtained in Token test, fluency for letters and working memory test. The results showed a general better performance in MCI-FTD patients as compared to both negative and positive PGRN mutations carriers (p < 0.01). No significant differences resulted between the comparison between negative and positive mutations PGRN carriers.

#### DISCUSSION

Our results show that FTLD patients carrying GRN mutations have different changes of EEG rhythms when compared to patients negative for GRN mutations. Specifically, GRN mutations carriers demonstrate an increment of the high alpha and a decreased of the theta power spectra. In this view, the presence of PGRN, which is a trophic factor for the nervous system, would allow a reorganization of the neural networks in a compensatory or adaptive way: accordingly, it has been reported that PGRN promotes neuronal survival and neurite outgrowth in cultured neurons (Van Damme et al., 2008) Likewise, under stress conditions it has been exhibited that PGRN improves neuronal survival (Kessenbrock et al., 2008). Data from GRN knockout experimental models suggest that PGRN deficiency leads to reduced synaptic connectivity and impaired plasticity, which may be contributing factors to FTLD pathology in human patients (Tapia et al., 2011; Xu et al., 2011). On the other hand, the white matter impairment in GRN mutation carriers has been directly proved in a recent study (Caroppo et al., 2014). In this study, four patients carrying GRN mutation were investigated and in all of them mostly confluent white matter lesions, affecting the periventricular subcortical white matter and U-fibers, were found mainly in the frontal and parietal lobes. This trophic activity could be shown by the maintenance of theta brain oscillations. Of note, fronto-parietal network are well recognized potential generators of theta oscillations (Beck et al., 2008; Ghetti et al., 2008; Whitwell et al., 2009), traveling on long pathway bundles of the white matter of fronto-parietal lobes and related subcortical structures like brainstem, and corpus callosum systems (Rohrer et al., 2010a,b). In particular, a recent study investigating the cortical generators of theta brain rhythm, through intracranial electrode recordings, have found that the higher percentage of cortical gated theta oscillations is situated in parieto-occipital regions (Raghavachari et al., 2006). So, a possible explanation of the spared maintenance of theta power in FTLD patients negative for GRN mutations as compared to GRN mutations carriers could be related to the relative integrity of parieto-occipital areas, embedded in the DMN.

Not surprisingly, in case of PGRN deficiency, the high alpha frequency spectral power increments. The alpha oscillation is the rhythm that mirrors the brain electrical signal of the parietaloccipital default system, profoundly connoted with higher cognitive abilities (Klimesch, 2012; Moretti, 2015a). Recent studies have shown that mutations in GRN are correlated with atrophy of the parietal lobes (Pickering-Brown et al., 2008; Seeley, 2008; Van Swieten and Heutink, 2008). In particular, a recent study investigating brain network connectivity with functional resting state MRI (Premi et al., 2014) utilizing three measures of both local and global connectivity (the regional homogeneity, the fractional amplitude of low frequency fluctuation and the degree centrality) has shown that parietal lobes are affected very early in GRN mutation carriers, so that the notion of fronto-parietal dementia for PGRN related disease should be considered. The parietal lobes are wellrecognized core regions of the DMN (Sala-Llonch et al., 2015). Previous studies have demonstrated that high alpha rhythm synchronization (or increase) is a marker of loss of function in the default system, as exhibited in patients with prodromal or overt AD (Moretti et al., 2011b, 2013a,b, 2014a,b; Moretti, 2015b,c,d). The increase of high alpha oscillation in FTD patients, characterized by the loss of PGRN, confirms the reliability of this biomarker as a sign of DMN impairment. Moreover, the lack of PGRN is confirmed to be connected to the disruptive of the default system, determining real reverberations on cognitive capacity.

As expected, MCI-FTD have better cognitive performance, especially in frontal function assessment tests, as compare to overt FTD patients both carriers and non-carriers of GRN mutation. Moreover, in MCI-FTD the lower spectral power of theta and alpha power could be explained with an initial stage of the disease in which EEG changes are not still clear. A possible fingerprint of this stage could be the ratio between alpha and theta power spectra. The progression of disease could modify the EEG oscillations, as observed in overt FTD and more strongly in GRN mutation positive patients. Anyway, future studies will clarify if the presence or absence of PGRN mutations also in MCI-FTD could unveil more peculiar brain oscillations changes.

#### Study Limitations

It should be remarked a main limitation of the study that is the small sample size. We want to highlight that it should be considered the exploratory nature of the study. In this view, the choice criteria and statistical outcomes are sufficiently robust to be confident in the overall reliability of the results. In particular, the straight difference between MCI-FTD and overt FTD patients confirm that results are not due to mere random chance results. Despite this limitation, it provides important results for future studies. Up to date, for the first time in this paper, the particular aspect of a neurophysiological EEG biomarker in GRN mutation carriers and non-carriers have been investigated. Most important, the detection of a neuropshysiological biomarker, related to functional, and structural changes, could open new windows on integrated research strategy about the molecular nexopathies shedding light on the way through which different proteinopathies could differently affect neural networks (Warren et al., 2013). Finally, the finding of a new biomarker could be helpful for early diagnosis, to monitor the progression of disease and to test disease-modifying drugs. Anyway, we are well aware

of the need to correlate the results with a wider morphological and neuropathological characterization as well as with other risk factors.

#### CONCLUSION

fnagi-08-00035 February 25, 2016 Time: 20:20 # 5

GRN mutations affect brain oscillatory activity. A better understanding of the complexity of PGRN biology in the brain will help guide the development of PGRN-modulating therapies for neurodegenerative disease. Further studies with a larger number of subjects are needed to confirm the present results.

#### AUTHOR CONTRIBUTIONS

DVM: Substantial contributions to the conception or design of the work. LB: substantial contribution for the

#### REFERENCES

acquisition, analysis, or interpretation of data for the work; and drafting the work. SF: substantial contribution for the acquisition, analysis, or interpretation of data for the work; AND drafting the work. MC: substantial contribution for the acquisition, analysis, or interpretation of data for the work; and drafting the work. GB: substantial contribution for the acquisition, analysis, or interpretation of data for the work; and drafting the work. RG: substantial contribution for the acquisition, analysis, or interpretation of data for the work; and drafting the work.

#### ACKNOWLEDGMENT

This work was supported by grants from Ricerca Corrente, Italian Ministry of Health.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2016 Moretti, Benussi, Fostinelli, Ciani, Binetti and Ghidoni. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.


*3.3. Characteristic Wavelengths*

S-G(7, 5)&

Fewer than 100 wavelengths were required to simplify the SCC evaluating models. The changing processes of RMSEV with different wavelength candidate subsets, which were chosen by SPA or MCUVE, are shown in Figure 3a,b, respectively. Using the combination of (SPA, PLSR) or (MCUVE, PLSR), characteristic wavelengths were selected (57 and 83, respectively). The minimum RMSEVs of SPA and MCUVE were 0.0692% and 0.0685%, respectively.

**Figure 3.** Changing processes of RMSEV with different wavelengths. (**a**) SPA; (**b**) MCUVE.

As shown in Figure 4a, 46 characteristic wavelengths distributed densely between 846.8 and 940.6 nm were obtained for the first selection method, while 10 characteristic wavelengths were scattered between 498 and 750 nm and 1 characteristic wavelength was located at 997.1 nm. Characteristic wavelengths selected by the second method were distributed mainly in the ranges of 757.7–796.7 nm, 828.3–847.5 nm, 866.8–910.3 nm, and 952.9–1006.9 nm, as shown in Figure 4b. The two methods both selected characteristic wavelengths in the range of 828.3–910.3 nm, which were correlated with the third overtone stretch of O-H and C-H functional groups.

**Figure 4.** Distribution of characteristic wavelengths. (**a**) SPA; (**b**) MCUVE.


**2. Oxidative stress and inflammation**

Nuts are sources of phenolic compounds and tocopherols with anti-inflammatory and antioxidant activities. The beneficial effects of cashew nuts on oxidative stress and acute and chronic inflammatory processes have recently been investigated in various in vivo experimental models on mice, such as ischemic injury, lung injury, pancreatic injury induced by cerulean, paw edema induced through carrageenan, osteoarthritis induced through monosodium iodoacetate (MIA), and colitis induced by dinitrobenzene sulfonic acid (DNBS) [37]. Antioxidative, anti-inflammatory, and analgesic activities of cashew nuts have been highlighted and hypothesized that these beneficial effects have correlation with the high phenolic contents in cashews mediating the 5-LOX COX pathway activation [37]. The effectiveness of Brazil nut and pistachio against inflammation and oxidative stress has also been demonstrated through *in vivo* studies [38].

Published study findings suggest that intake of nuts may contribute in the protection against inflammatory processes and oxidative stress, which is mainly because of the abundantly present bioactive metabolites such as carotenoids, flavonoids, and phenolic compounds found in the kernel and skin as reported in published studies.

Clinical and epidemiological studies suggested that antioxidant vitamins, ω-3 PUFA, magnesium, L-arginine, and dietary fiber may play an essential role in the modulation of inflammation. The relationship observed between inflammatory markers and frequent nut consumption has reported mixed results when investigated in controlled feeding intervention [39].

#### **3. Prevention of cardiovascular diseases**

Association between nut consumption and decreased risk of ischemic heart disease (IHD), cardiovascular disease (CVD), and the incidence and mortality of CVD has been consistently proven by the epidemiological studies [40]. Consumption of nuts also increases longevity in addition to protection against CVD [41]. Nuts being the dense source of energy due to the high content of bioactives play significant role in decreasing the risk of CVD through multiple mechanisms: by having positive influence on lipid and/or glucose homeostasis [42], hypercholesterolemia [43], obesity [44], oxidative and inflammatory biomarkers, and antioxidant defenses.

Nuts consumption has positive effects on various CVD risk factors including improvements in lipoprotein cholesterol, total cholesterol (TC), and triglycerides [45]. The authors of the published studied attributed that cardioprotective potential of almonds is due to their dense bioactive contents such as phytosterols, dietary fiber, and high poly-unsaturated fatty acids contribute to their conserving HDL-C and LDL-C lowering effects [46]. Later on, it was reported that almonds incorporated [47]. Additionally, walnut supplementation increases the HDL-C after treatment for 6 months possibly due to the higher intake of PUFA.

The reduced risk of CVD through almond snacking by increasing variation in heart rate in healthy adults during mental stress and the consumption of whole almond was linked with better quality diet and reduced CVD risks in the adult population of UK have been demonstrated in a randomized controlled trial study [48]. The authors also reported the association of almond snacks consumption with significant endothelial function improvement, in addition to the lowering LDL-C among adults with above average risk of CVD.

Numerous studies have indicated that eating nuts reduces the risk of heart disease and stroke, due to the fact that they contain polyphenol compounds that reduce the level of bad cholesterol and work to reduce the rate of inflammation in the body by reducing inflammatory markers C-reactive protein (CRP) and interleukin 6 (IL-6) [49].

Also, in one study of five people who took cashews, lower blood pressure was observed in addition to lower triglyceride levels [50].


**2. Materials and Methods**

#### *2.1. Plant Materials*

The research material (berries) used in this study was collected from the vineyard at the Zhengzhou fruit research institute (China) during 2020. The varieties used in the present research were 'Italia', 'Benitaka', 'Muscat of Alexandria', 'Flame Muscat', 'Rosario Bianco', and 'Rosario Rosso'. The vines were 10 years old with 'Y'-shaped tree forms. The berries of each cultivar were in the véraison stage, from 10 wpf (weeks post flowering) to 11 wpf. The red varieties showed notable change in berry color at 11 wpf (Figure 2). Three berries from the upper, middle, and lower parts of each cluster were selected from six uniform clusters. The berry skins were peeled off quickly and frozen in liquid nitrogen immediately. All frozen samples were stored at −80 ◦C for further analysis.

All the samples were allotted numbers as follows: It10 ('Italia' 10 wpf berries), It11 ('Italia' 11 wpf berries), Be10 ('Benitaka' 10 wpf berries), Be11 ('Benitaka' 11 wpf berries), Ma10 ('Muscat of Alexandria' 10 wpf berries), Ma11 ('Muscat of Alexandria' 11 wpf berries), Fm10 ('Flame Muscat' 10 wpf berries), Fm11 ('Flame Muscat' 11 wpf berries), Rb10 ('Rosario Bianco' 10 wpf berries), Rb11 ('Rosario Bianco' 11 wpf berries), Rb11 ('Rosario Rosso' 10 wpf berries), and Rr11 ('Rosario Rosso' 11 wpf berries) (as shown in Figure 2, respectively).


*6.1. Purinergic Receptors*

In acute and chronic pain models, small- and medium-diameter sensory neurons, which express transient receptor vanilloid-1 (TRPV1) channels and/or adenosine triphosphate (ATP)-gated P2X3 receptors, are the important pain transducers of noxious stimuli [57]. In women with EM, TRPV1 receptor expression has been demonstrated to be elevated in endometriotic lesions and correlated with pain [36,37,57].

Purinergic receptors are ligand-gated ion channels that are expressed in sensory neurons which can be activated by adenosine triphosphate (ATP). P2X3 is one such purinergic receptor. In women with EM, ATP released during retrograde menstruation and due to mechanical stretch from endometriotic adhesions or fibrotic scar tissue could potentially activate P2X3 receptors, leading to neuronal hypersensitivity and pain [58]. Indeed, its expression in the endometrium of women with EM was significantly higher compared to control endometrium and interestingly was also correlated with the severity of pain reported [58,59]. Current interest in targeting P2X3 in EM is high, with multiple pharmaceutical companies having initiated drug programs, one of which has begun to enter Phase IIb clinical trials (NCT04614246).