Reproductive Management in Sheep

#### **Chapter 5**

## Reproductive Rates of Merino Ewes and Offspring Quality under AI Program

*Edward Narayan, Gregory Sawyer, Natalie Hoskins and Greg Curren*

#### **Abstract**

Reproductive wastage is a major economic burden in sheep production globally, especially within Australia as livestock production systems face increased pressure from climatic variability (e.g. prolonged droughts or flooding). Sheep are sensitive to acute changes in their environment such as heat stress, which if not adequately monitored will result in significant production losses such as reproductive failure, increased parasite and worm burden, morbidity and mortality risks. Through basic and applied research in the areas of stress and reproductive physiology our team has made significant advancements in the understanding of sheep behaviour and physiological responses to acute and chronic stressors. Using minimally invasive hormone monitoring technology in combination with field based assessment of sheep health and productivity traits, our team has delivered new knowledge on how sheep react to acute environmental stress and how it impacts on sheep reproduction. In this chapter, we evaluated the fertility rates and embryo quality of Merino ewes under AI breeding program. We discuss factors such as heat stress that can impact on ewe and offspring quality.

**Keywords:** Reproductive Wastage, Merino Sheep, Heat Stress, Resilience, Embryo Quality

#### **1. Introduction**

Merino ewes account for 75% of all breeding ewes in Australia, comprising of those used for high quality fleece production, as well as for the production of first cross lambs for the meat market [1]. Despite Australia's high usage of this breed, the reproductive performance of merinos is often described as being substantially lower than those of other breeds [2–4]. Many studies have attempted to understand the sources of this poor performance, in the hope that by better understanding the causes of the loss, that they can better implement strategies to mitigate the losses.

Kilminster and Greeff [5] found that significantly lower rates of conception were found in Merino ewes who were joined at only 8 and 9 months of age, when compared to their same age Dorper and Damara counterparts. Ewes that were maiden, that is, having their first lambs, were also more likely to have lower success rates [3, 6]. In these studies, this conception rate drastically increased as the

ewes matured and became experienced mothers. A study [7] into the reason for the increasing conception rate with age and amount of parturitions found that the rate of cervical passage, as is used for transcervical artificial insemination, increased with increasing parturitions due to cervical stretching. This study did not observe ewes beyond 3 parturitions so did not observe if this cervical stretching may cause problems into older age. A 2015 study [8] of 22,758 Churra ewes, a Spanish dairy sheep breed, reported that ewes are most fertile between 1.5 and 4.5 years of age. Ewes older than 4.5 years often would have had numerous parturitions, a factor that Anel determined was a contributing factor to the decline of fertility.

Ewes that conceive multiple foetuses experience a compounding risk of poor reproductive performance, as there is a high risk of losing one foetus between pregnancy scanning and parturition. Those that are able to keep both foetuses until parturition are still at risk of losing a lamb during the sensitive post-parturition period [3, 9].

Sheep are considered to be seasonal breeders. They experience marked changes in their behaviour, endocrine and ovulatory levels between their ovulatory and anovulatory seasons. Hormonally, luteinizing hormone (LH) remains at a relatively constant level, although the pulsation rate is lowered. Plasma progesterone is essentially undetectable during anoestrus [10]. Farmers are able to manipulate this seasonal variation through the use of light pulses and melatonin supplementation to create short days and long days [11]. Oestrus is stimulated after the longest day of the year, at which point the day-length begins to shorten. In Australia, this is late December. Fogarty (year) found that oestrus and fertility peaked during autumn (February) joining with the lowest fertility rate being seen in Spring (October and November) joining's.

The impact of the thermal environment on fertility and conception rate is not a new concept. In 1964, Dutt placed ewes in a temperature-controlled room prior to and/or after breeding and studied their responses [12]. The results showed a significantly lower rate of fertilisation in the heat-treated ewes. Placental research has similarly shown that high ambient temperatures reduce success rates. Bell et al. [13] and Early et al. [14] found that placental development is reduced by up to 54% in heat-treated ewes, which had a carry-on effect to the offspring: as the placenta was smaller, the foetus's growth was stunted. Studies by Kleeman et al. [3, 15] found that fertility was negatively influenced by the number of days ambient temperatures were above 32.0°C during mating, suggesting that high ambient temperatures may reduce embryo survival.

In spite of this large body of research into reproductive wastage from the perspective of gestational or pre-weaning loss, as well as genetic quality concerns, there is limited research into the impact of the ewe on the quality of her offspring, especially long-term studies that look beyond weaning and into adulthood. This study aims to fill this gap by determining factors in the ewe that will lower her reproductive success and the quality of offspring using lifetime production data from the MerinoLink Limited Sire Evaluation Program.

#### **2. Methods**

#### **2.1 MerinoLink limited sire evaluation program**

The MerinoLink Limited Sire Evaluation Program is designed as a standard sire evaluation trial that follows progeny of selected Merino Superior Sires (MSS), assessing their characteristics at 10 months and 22 months of age, in line with the Australian Merino Sire Evaluation Association. The selected traits are those deemed to be of value to breeders and commercial producers. For the purpose of our study, we do not know which progeny belong to which sire, as this was deemed a potentially confounding variable if sire identification was known.

#### **2.2 Study group**

The 2016 breeding season was conducted at a commercial farming property in Jugiong, NSW (−34.770150, 148.304470). Ewes were managed as one contemporary flock until 10 days before lambing, when they were separated into their respective sire groups. This was to ensure that there was no external influence by environment or pasture type and quality. The ewes were also given equal opportunity and access to a supplementary feeding program. The feeding program was designed to sure that nutritional requirements were met throughout all stages of gestation. The researchers were supplied with the following data from the research data providers; it is thus mixed aged ages spread evenly across all sire groups. The exact age grouping for each sire is unknown. The foundation ewes that were used to generate the 2016 and 2017 drops were sourced from five flocks and allocated evenly across all sire groups, the foundation ewe base consisted of:

Insemination occurred in a shed environment on the 23rd February 2016 with a total of 107 ewes each being inseminated *via* artificial insemination to one of twelve randomly selected sires. The insemination day ran for a total of 5 hours and 22 minutes, from 10:48:19 to 16:10:23. Breaks were had at 11:48 for 41 minutes, 13:11 for 16 minutes and 14:01 for 1 hour and 8 minutes. All sires were previously evaluated for semen quality. Each ram was given 50 mixed aged ewes as noted above. In the 2016 trial the age breakdown of the sires used were 2011 – 1, 2012 – 3, 2013 – 3, 2014 – 3, 2015 – 5; 2017 trial 2011 -1, 2013 – 3, 2014 – 4, 2015 – 5. This was from a sire evaluation trial and various sires semen was used from various studs.

At time of the research being conducted the standard AI protocol in Australia based on AllStock Artificial Breeding Services (www.allstock.com.au) was implemented. All laparoscopic AI procedures conducted on the ewes were performed by the same qualified AI technician. On the day of AI, ewes were sedated with 0.05 mg/kg of Zylazil injection 20 minutes prior to the AI procedure. Ewes were then artificially inseminated via laparoscopy, with frozen semen. Semen quality was assessed post thawing by a qualified veterinary surgeon. Rectal temperature was recorded twice (immediately before sedation and 30 min post AI). All laparoscopic AI procedures conducted on ewes in this study were performed by a qualified veterinary surgeon.

Pregnancy scanning occurred on the 26th May 2016, where ewes were scanned as being dry (not pregnant), pregnant with a single lamb or pregnant with twins. Ewes gave birth between the 22nd and 30th July, lambs were marked on the 2nd September 2016, and weaning occurred on the 29th October 2016. The data supplied to the researchers was from an Australian Industry trial that the researchers had no influence on the methodology or overall design. Authors of this paper understand that pregnancy can be detected post 45 days in sheep and would recommend this to be of best practice. However, it is believed that 92 days would have allowed back up rams to inseminate ewes that had lost a lamb early after insemination and be drafted off from the trial mob with small embryos. All ewes are commercial ewes were being used in the trial.

The 2017 breeding season was conducted at a property south of Yass NSW (−34.977260, 148.855810). A total of 800 ewes were managed in the same manner as the 2016 flock, until 10 days before parturition, when they were divided into 5 mobs. Data sets for 531 individuals were made available for our study, of which 136 were randomly selected to be used for further data collection and analysis.

Insemination occurred in a shed environment on the 28th February and 1st March 2017, where the 800 ewes underwent artificial insemination to one of sixteen sires, selected at random. The first day began at 7:51:30 and ran to 14:32:51 with breaks at 9:08 (37 minutes) and 11:38 (66 minutes). The second day began at 7:18:03 and finished at 13:06:40, with breaks at 9:06 (47 minutes) and 11:38 (65 minutes).

The 2017 flock were scanned for pregnancy confirmation on the 24th May 2017. All ewes gave birth between 28th July and 8th August 2017. Marking occurred on the 1st September 2017, where their sex was recorded and lambs were weaned from the ewes on the 9th November 2017. At the time of weaning, the data for the category "weaning weights" was collected. Post-weaning weights were collected when the lambs were 6.5 months old (14th February 2018), and yearling weights were taken when the lambs were 12.5 months old (27th June 2018). Finally, fleece data, including fibre diameter, staple length, staple strength and wool weight (greasy and clean) were obtained at 10 months of age, on the 21st May 2018.

For the 136 selected for further analysis (study group), we obtained data on the year of birth of mother, diagnosis as twin or singleton at the time of pregnancy ultrasound and the body temperature (rectal) of mother at insemination.

#### **2.3 Types of data collected**

For the purpose of this data set, lambs considered to be of the "weaning" age category were between 6 weeks and 4 months of age (42 to 120 days). Those in the "post-weaning" category were between 4 and 10 months of age (120 to 300 days). Finally, those in the "yearling" category were between 10 and 13 months of age (300 to 400 days).

Fibre diameter refers to the measurement in micrometres (microns) of the wool fibres from an individual sheep. Merino sheep are a breed specifically designed for their fine wool diameter, aiming for 20 microns or lower; a lower micron size denotes a finer wool, and thus a higher quality wool. Staple length is the length in millimetres of a piece (staple) of wool. The length of a staple determines its end use – whether it will be used for weaving or knitting. Staple strength refers to the amount of force required to break a wool staple, recorded as Newtons per kilotex

#### *Reproductive Rates of Merino Ewes and Offspring Quality under AI Program DOI: http://dx.doi.org/10.5772/intechopen.99617*

(Nkt). A kilotex refers to a staple of a given thickness. This informs us of the efficiency of wool processing; how likely the fleece is to break during processing.

All fleece, as shorn straight from the sheep, including skirtings is called greasy fleece weight. As this weight occurs before cleaning, it includes all fibre, vegetable matter, dirt, wax and other environmental contaminants. Clean fleece weight is the weight after these contaminants have been removed, and the fleece has been washed. It is calculated using the formula:

Clean fleece weight kg greasy fleece weigh ( ) = t kg x washing yield % ( ) ( )

#### **2.4 Statistical analysis**

All data was analysed using IBM SPSS Modeller (SPSS Inc. 1994). A covariate analysis and chi square analysis were performed for both the 2016 and 2017 flocks to analyse the fertility rates for time of day of insemination. The 2017 flock underwent further analysis, including regression analysis of bodyweight, greasy and clean fleece weights, yearling fibre diameter and temperature at insemination. One sided T-tests were used to assess yearling staple strength, staple length, ewe body temperature at insemination vs. progeny sex and body weight at weaning. Year of birth of mother was analysed using a Fishers one-sided exact test. Differences were considered significant at P < 0.05.

Conception rate (also referred to as "artificial insemination rate" or "fertility rate") is calculated using the formula: conception rate = (successful conceptions)/ (total ewes inseminated)

#### **3. Results**

#### **3.1 Fertility in 2016 flock**

The 2016 flock (n = 107) had an artificial insemination success rate of 55% (n = 59), with a further 40 ewes conceiving via a backup ram after artificial insemination. This combined total lead to a total fertility rate of 93% (n = 99).

The earliest period of the day (period 1) had the most conceptions, with 40.67% (n = 24) of all successful inseminations occurring in this period. This was a rate of 73% of the period 1 ewes that conceived. The lowest period in the day was period 2 with 11.86% of all conceptions occurring in this period. This was a rate of 29% of the period 2 ewes that conceived; this different is statistically significant

#### **Table 1.**

*Fertility rates of merino study ewes (n = 107) via artificial insemination in 2016 by period of day inseminated. Singleton vs. twin rates provided based on scanning data for those that conceived.*

(Fishers one-sided; P = 0.001). However, as the day progressed, the conception rate improved, with 32% (n = 19) of all conceptions, 61% of the period's conceptions, occurring in period 4 (**Table 1**). The difference in conception rate between period's 2 and 4 was statistically significant (Fishers one-sided; p = 0.018). The difference between period 1 and 4, 2 and 3, and 4 and 3 were not statistically significant (Fishers one-sided; p = 0.252).

Despite this lower rate of conception in period 2, this period produced a higher rate of lambs, with six of the seven period 2 ewes having twins. The proportions of twins and singles in the other three periods (periods 1, 3 and 4) were all similar and there was a borderline statistically significant difference between period 2 and all other periods (Fishers one-sided; p = 0.082).

#### **3.2 Fertility in 2017 flock**

The 2017 flock had a total conception rate of 87% (n = 180 lambs at scanning). 66% (n = 138) of these conceptions occurred via artificial insemination, with the remainder of lambs being conceived via the use of a back up ram (n = 42 lambs at scanning). Only 28 ewes failed to conceive during this period via either artificial insemination or after spending two cycles with a back-up ram.

In 2017, the artificial insemination was performed over three periods. 36.9% (n = 51 lambs at scanning) of all lambs conceived, were from ewes inseminated in the first period. The within-period conception rate was 70.8%. The middle period (period 2) contributed the highest conception rate of the day, with 45.6% (n = 63 lambs at scanning) of all lambs conceived on the day, with a within-period success rate of 77.7%. The final period (period 3) had the lowest conception rate, with 17.3% (n = 24%) of lambs being conceived in this period. The ewes that were inseminated in the afternoon period (period 3), including those that failed to conceive *via* artificial insemination, but that went on to conceive via the use of a back-up ram had the highest twinning rate, with 55% of lambs in this period scanning as twins. This was followed closely by the middle period (period 2) which had a twinning rate of 39%, followed by period 1 which had a twinning rate of 49%. The difference between the periods of insemination performed before the major break of the day (periods 1 and 2) and the afternoon period (period 3) was statistically significant (Chi2; p = 0.0005) (**Table 2**).

#### **3.3 Year of birth of mother**

In 2017, both the eldest ewes (born in 2011) and the youngest ewes (born in 2014) each contributed 13% (n = 24 each) of lambs in the 2017 conceptions. Both

#### **Table 2.**

*Fertility rates of merino study ewes (n = 136) via artificial insemination and back-up ram in 2017 including the rate of singleton and twins from both the artificial insemination and ram back up usage.*

*Reproductive Rates of Merino Ewes and Offspring Quality under AI Program DOI: http://dx.doi.org/10.5772/intechopen.99617*

**Table 3.**

*The effect of year of birth of mother on the conception of single or twin pregnancies.*

of these also had a twinning rate of 46% (n = 11 each). The most fertile ewes were those born in 2012, with 31.7% (n = 57) of lambs conceived by these ewes, of which 63% (n = 36) were twins. The 2013 born ewes contributed 20% (n = 36) of lambs in 2017, with 48% (n = 36) being twins. The difference in fertility between 2012 ewes and all other ewes was statistically significant (Fishers one-sided; p = 0.033), however 2011, 2013 and 2014 ewes were not significantly different from each other (**Table 3**).

#### **3.4 Temperature at conception (2017)**

The distribution of maternal body temperature at time of conception was slightly skewed from normal (**Figure 1**). The ewes body temperatures (rectal) ranged from 39.0°C to 40.9°C, with a mean of 39.78°C, and a similar median of 39.7°C. We considered temperatures over 40.2°C to be abnormal, as only 11% (n = 15) had temperatures at or over this point. The variation in body temperatures did not produce different rates of male or female progeny (t-test; p = 0.021).

Regression analysis showed no statistical significance for temperature\*all\_ weaning\_weight (p = 0.506), temperature\*postweaning\_weight (p = 0.215), temperature\*male\_weaning\_weight (p = 0.783), and temperature\*male\_postweaning\_weight (p = 0.532). Temperature\*female\_weaning\_weight was not significant, but had a lower p-value than the other weaning samples (p = 0.281) and

#### **Figure 1.**

*Histogram of temperature of 2017 ewes at conception. Mean = 39.78°C. temperatures over 40.2°C were considered abnormal.*

temperature\*female\_postweaning\_weight was significant (p = 0.021). No regression was performed for yearling weights.

Conception temperatures above and below 39.5°C had a borderline significance for female progeny (t-test; p = 0.13), with a difference in post-weaning weight of +0.46 kg. Conception temperatures above and below 40.2°C likewise showed statistical significance (t-test; p = 0.033) with a difference in post-weaning weights of +1.09 kg.

Weaning weights for female progeny likewise showed non-significance (t-test one-sided; p = 0.281), with a difference in average weaning weight for both low (<40.2°C) and high (>40.2°C) maternal conception temperature of +0.41 kg (low = +0.06 kg; high = −0.36 kg). Finally, yearling weights showed significance, for conception temperatures above and below 39.5°C (±1.9°C) (t-test; p = 0.0300) and above and below 40.2°C (t-test; p = 0.0073).

#### **3.5 Pregnancy scans (2017)**

At the time of scanning, there was a 1:1 ratio of singleton and twin lambs, with 54 ewes scanned as being pregnant with singletons and a further 54 scanning as having twins. Of these 54 twin-scanned ewes, 48% (n = 26) lost one of their lambs, having only a single survive to birth.

#### **3.6 Progeny sex (2017)**

In the study group, the ratio of males to females was 0.84:1, with 62 males and 74 females being born.

#### **3.7 Progeny weight (2017)**

Regression analysis found no significant difference in each age category (weaning, post-weaning and yearling) between the study and non-study groups. As yearlings, however, there were proportionately more progeny in the study group that were under the mean weight than those in the non-study group, who showed a higher range of weights (**Figure 2**).

The change in weight between age categories was not statistically significant through regression analysis, however the range of weight changes is notable – some sheep gained weight between age groups, whilst others lost weight between ages. The widest range of weight changes, including loss of weight, occurred from weaning to yearling age (**Figure 3**).

#### **3.8 Fibre diameter, length and strength (2017)**

There was no statistically significant difference (t-test; p = 0.12) in yearling fibre diameter (YFD) between the study and non-study groups. The study group averaged +0.073, whilst the non-study group averaged −0.006. A regression analysis showed no relationship between yearling fibre diameter with the mother's temperature, year of birth, or the sex of the progeny tested. There was, however, a highly significant relationship with whether the progeny was scanned as a twin or singleton, and its yearling fibre diameter. Those scanned as twins had an average YFD of −0.084, and those scanned as single lambs averaged at +0.398.

The final yearling fibre diameter analysis was a regression of yearling fibre diameter versus scan fecundity (single or twins) and sex. This found that the sex\*fecundity interaction was not significant, nor the sex\*twinning interaction, but *Reproductive Rates of Merino Ewes and Offspring Quality under AI Program DOI: http://dx.doi.org/10.5772/intechopen.99617*

#### **Figure 2.**

*Boxplots of bodyweights at weaning (wwt = blue), post-weaning (pwt = red) and yearling (ywt = green) for study and non-study groups (study group = 0; non-study group = 1).*

#### **Figure 3.**

*Boxplots of change in bodyweights in study (0) and non-study (1) progeny. Pw\_w (blue) = weaning to post-weaning; y\_pw (red) = post-weaning to yearling; y\_w (green) = weaning to yearling.*

was the for sex\*singletons. Females had a mean yearling fibre diameter of +0.057, and males had a mean diameter of +0.524. The singles showed a marked bimodal distribution, but not those born as twins (**Figure 4**).

#### **3.9 Staple length and staple strength**

Yearling staple strength was significantly greater in the study group (t-test; one sided p = 0.032) with the study group's mean staple strength at +0.0621, and

**Figure 4.** *Histogram of yearling fibre diameter by scan fecundity (1 = singletons, 2 = twins).*

**Figure 5.**

*Boxplots of yearling staple length (ysl = blue) and yearling staple strength (yss = red).*

the non-study group's mean at +0.033 (**Figure 5**). Yearling staple length was not significantly different between study and non-study groups (**Figure 5**).

#### **4. Discussion**

The MerinoLink program with its comprehensive range of collected data has been a powerful tool for understanding the relationship the ewe has with her progeny. In the present study, we found that the youngest ewes (born in 2014) and

#### *Reproductive Rates of Merino Ewes and Offspring Quality under AI Program DOI: http://dx.doi.org/10.5772/intechopen.99617*

the eldest ewes (born in 2011) had the lowest conception rates (13% each). The low rate of conception from young ewes is consistent with data presented by Kleeman and Walker [3, 15] who found that maiden Merino ewes had a fecundity rate up to 11% lower than the mature ewes of their study. Additionally, an Egyptian study by Abdel-Mageed et al. [6] found that maiden ewes (of the Rahmani and Barki breeds) had a reproductive wastage rate as high as 70% in maiden ewes (vs. 42% for mature ewes). Mature ewes in both of these studies had higher successful conceptions and lower reproductive wastage, as was seen in our study with the 2012 and 2013 ewes (31.7% and 20% respectively). The ewes born in 2012 also had a significantly higher twinning rate with 63% of lambs born as twins. Kleeman and Walker [3, 15] reported a similar result and found that this was due to a higher ovulation rate with more multiple ovulations, as is often required for twins to be conceived.

The time of day of conception was an important factor for the overall conception rate as well as the rate of singletons and twins being conceived. The 2016 flock had the highest conception rate in the first period of the day (73% success), followed by the last period of the day (61% success). The middle two periods experienced relatively low rates of conception (29% and 47% respectively). Despite this low rate of conception in the middle periods, period 2 experienced the highest rate of twins conceived, with 6 out of 7 (86%) of ewes conceiving twins. Unlike the 2016 flock, the 2017 flock saw the highest conception rate in the middle period (45.6% of all AI conceptions), followed by the first period (36.9% of AI conceptions). The final period of the day had only a 17.3% conception rate from AI. Overall, the 2017 flock had a conception rate 11% higher than 2016 (2017 = 66%, 2016 = 55%), and was 5.9°C cooler than the 2016 insemination day.

Previous studies suggest two potential lines of reasoning for this daily variation. The first potential line is that of daily melatonin variation. Melatonin, synthesised and secreted from the pineal gland is the hormone responsible for seasonal fertility in sheep, and is regulated by the day-night cycle. Seasonally, the study ewes were in prime fertility, however daily melatonin variations are rarely considered as a potential fertility factor. Melatonin implants [16–19] have been shown to not only prolong the breeding season, but also to improve the proportion of successful conceptions and the fecundity rate. Melatonin has been identified as a component required for in vivo oocyte maturation, as it has been quantified in the granulosa cells of healthy oocytes (Tamura et al., 2012, as cited in [20]). Tamura et al. and Peris-Frau et al. (2017, as cited in [20]) also suggested that melatonin may act as an antioxidant in the oocyte follicle, protecting it from reactive oxygen species (ROS), which are known to cause damage to oocyte and granulosa cells. Peris-Frau's studies added melatonin to the collecting media during ovary transport and found that the rate of degradation declined significantly. Whilst we did not measure melatonin levels in our study ewes, previous studies of daily melatonin changes indicate that melatonin is at its highest concentration early in the morning, declining until the evening when an animal returns to sleep, at which point the levels can be replenished.

The second line of reasoning involves the ambient and ewe temperatures. Decades of studies have shown that ewes have far higher conception rates when not exposed to high ambient temperatures and heat stress. Our present study did not have a heat treatment or comparison to cooler days, with all ewes being inseminated on the same day. We did, however, take rectal temperatures at the time of insemination, which allowed us to observe the temperature of ewes. There are many different numbers given for the "normal" rectal temperature of a sheep, with some suggesting as low as 38.3°C [21, 22]. The Australian Veterinary Association [23] suggests that a normal resting rectal temperature should be approximately 39°C, with mild heat stress beginning at 39.5°C. In the present study, the rectal temperature had a mean of 39.78°C, ranging from 39.0°C to 40.9°C. No ewes were reported as having

low rectal temperatures and temperatures over 40.2°C were considered abnormal, with only 11% having temperatures over this range. The 11% of ewes with temperatures over 40.2°C are considered by the AVA to be in moderate to severe heat stress. Although the ewes were inseminated in a shed condition, the shed undergoes natural temperature variations throughout the day, potentially explaining this ewe temperature variation, and thus conception rate.

The biggest impact that abnormally high temperatures had on our study group was that female progeny were born significantly smaller and remained smaller even into yearling age. This has a definite potential impact for the merino industry, both in terms of available surface area for which to grow fleece, for capacity to carry lambs, and even fertility. There appears to be limited research about as to the potential reasons for this. One study considered the effect of heat on the placenta [13, 14]. This study found that high heat exposure caused placental stunting of up to 54%. They found that placental RNA and DNA content were reduced as were maternal plasma concentrations of progesterone, cortisol and placental lactogen. However, if this was the cause of smaller females, we would expect to see this in the male progeny as well.

Unfortunately our study had several confounding variables that we could not eliminate. Specific data relating to ram usage over each ewe was unavailable due to the sensitivity of that information, but this meant we were unable to consider the impact of the ram itself. A 2005 paper by Anel et al. studied the potential factors influencing the success or failure of artificial insemination. Overall, they found that the year, season and the technique of AI were the most important factors that would predict success or failure of insemination. Therefore, we suggest that future studies should consider minimising these confounding variables to ensure consistency and accuracy.

Based on our results, we conclude that the lifetime data program can be a highly effective tool to understand the impact of the parents on the progeny, both in terms of genetic variation and environmental factors. Future studies should consider using this method to observe a wide variety of factors including pre-conception, post-conception, throughout gestation and into the adulthood of the progeny. Further research is also required to better understand the link between abnormally high rectal temperatures at conception and the overall size of female progeny.

#### **Acknowledgements**

NS conducted field project research under the principal supervision of EN and co-supervision of GS. Statistical analysis was performed by GC. We thank the Australian Merino Sire Evaluation Site and the MerinoLink Group for the retrospective analysis of their 2016-2017 data. We thank two anonymous reviewers for peer review. The funding for this research was provided by New South Wales Stud Merino Breeders (NSWSMB) association, Australian Wool Innovation (AWI), and Australian Wool Education Trust (AWET).

*Reproductive Rates of Merino Ewes and Offspring Quality under AI Program DOI: http://dx.doi.org/10.5772/intechopen.99617*

#### **Author details**

Edward Narayan1,2,3\*, Gregory Sawyer4 , Natalie Hoskins3 and Greg Curren3

1 Faculty of Science, School of Agriculture and Food Sciences, The University of Queensland, St. Lucia, Australia

2 Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St. Lucia, Australia

3 School of Science, Western Sydney University, Penrith, Australia

4 Faculty of Science, School of Life and Environmental Sciences, The University of Sydney, Sydney, Australia

\*Address all correspondence to<EMAIL_ADDRESS>
© 2021 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.

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[7] Windsor, D. (1995). Factors influencing the success of transcervical insemination in Merino ewes. Theriogenology, *43*(6), 1009-1018. doi: 10.1016/0093-691x(95)00065-g

[8] Anel, L., Kaabi, M., Abroug, B., Alvarez, M., Anel, E., & Boixo, J. et al. (2005). Factors influencing the success of vaginal and laparoscopic artificial insemination in churra ewes: a field assay. Theriogenology, *63*(4), 1235-1247. doi: 10.1016/j. theriogenology.2004.07.001

[9] Hatcher, S., Atkins, K., & Safari, E. (2009). Phenotypic aspects of lamb survival in Australian Merino sheep. Journal Of Animal Science, *87*(9), 2781-2790. doi: 10.2527/jas.2008-1547

[10] Scaramuzzi, R., & Baird, D. (1977). Pulsatile Release of Luteinizing Hormone and the Secretion of Ovarian Steroids in Sheep During Anestrus. Endocrinology, *101*(6), 1801-1806. doi: 10.1210/endo-101-6-1801

[11] Chemineau, P., Malpaux, B., Delgadillo, J., Guérin, Y., Ravault, J., Thimonier, J., & Pelletier, J. (1992). Control of sheep and goat reproduction: Use of light and melatonin. Animal Reproduction Science, *30*(1-3), 157-184. doi: 10.1016/0378-4320(92)90010-b

[12] Dutt, R. H. (1964). Detrimental effects of high ambient temperature on fertility and early embryo survival in sheep. International Journal of Biometeorology*, 8*(1), 47-56. Retrieved from https://link.springer.com/ article/10.1007/BF02186927

[13] Bell, A., McBride, B., Slepetis, R., Early, R., & Currie, W. (1989). Chronic Heat Stress and Prenatal Development in Sheep: I. Conceptus Growth and Maternal Plasma Hormones and Metabolites. *Journal Of Animal Science*, *67*(12), 3289. doi: 10.2527/ jas1989.67123289x

[14] Early, R., McBride, B., Vatnick, I., & Bell, A. (1991). Chronic heat stress and

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prenatal development in sheep: II. Placental cellularity and metabolism. Journal Of Animal Science, *69*(9), 3610-3616. doi: 10.2527/1991.6993610x

[15] Kleemann, D., & Walker, S. (2005b). Fertility in South Australian commercial Merino flocks: sources of reproductive wastage. Theriogenology, *63*(8), 2075-2088. doi: 10.1016/j. theriogenology.2004.06.017

[16] Abecia, J. A., Palacín, I., Forcada, F., & Valares, J. A. (2006). The effect of melatonin treatment on the ovarian response of ewes to the ram effect. Domestic Animal Endocrinology*, 31*(1), 52-62. doi:10.1016/j. domaniend.2005.09.003

[17] Cevik, M., Yilmazer, C., & Kocyigit, A. (2017). Effects of melatonin implantation on the fertility potentials of kivircik and charollais ewes and rams during the non-breeding season. Polish Journal of Veterinary Sciences*, 20*(3), 501-506. doi:10.1515/pjvs-2017-0060

[18] Mura, M. C., Luridiana, S., Farci, F., Di Stefano, M. V., Daga, C., Pulinas, L., Carcangiu, V. (2017). Melatonin treatment in winter and spring and reproductive recovery in sarda breed sheep. Animal Reproduction Science*, 185*, 104-108. doi:10.1016/j. anireprosci.2017.08.009

[19] Yilmazer, C., Cevik, M., & Kocyigit, A. (2018). Effects of subcutaneous melatonin implants and short-term intravaginal progestagen treatments on estrus induction and fertility of kivircik ewes on seasonal anestrus. *Polish Journal of Veterinary Sciences, 21*(2), 353-359. doi:10.24425/122604

[20] Abecia, J., Forcada, F., Vázquez, M., Muiño-Blanco, T., Cebrián-Pérez, J., Pérez-Pe, R., & Casao, A. (2019). Role of melatonin on embryo viability in sheep. *Reproduction, Fertility And Development*, *31*(1), 82. doi: 10.1071/rd18308

[21] Fielder, S. (2019a). Normal Rectal Temperature Ranges. Retrieved 11 November 2019, from https://www. msdvetmanual.com/special-subjects/ reference-guides/ normal-rectal-temperature-ranges

[22] Fielder, S. (2019b). Normal Rectal Temperature Ranges. Retrieved 11 November 2019, from https://www. msdvetmanual.com/special-subjects/ reference-guides/ normal-rectal-temperature-ranges

[23] Australian Veterinary Association Ltd. (2018). *Heat Stress Risk Assessment (HotStuff): Issues Paper* [Ebook] (p. 6). Australian Veterinary Association Ltd. Retrieved from http://www.agriculture. gov.au/SiteCollectionDocuments/ biosecurity/export/live-animals/ australian-veterinary-association.pdf

#### **Chapter 6**

## Ovine Artificial Insemination in the Maghreb Region: Present Status and Future Prospects

*Moufida Atigui and Mohamed Chniter*

#### **Abstract**

Artificial insemination (AI) plays a key role in the genetic improvement of farm animals. Although it is widely used for cattle in the Maghreb region, it is scarcely applied in sheep at farm level. This is not only due to low fertility and irregular results that range between 30 to less than 76% for both cervical AI with fresh semen and laparoscopic insemination with frozen semen in most of studied breeds and also because of low results related to conditioning of fresh, chilled and frozen rams′ semen. An appropriately literature analysis was conducted to highlight the importance of sheep breeding in the Maghreb region particularly in Morocco, Algeria and Tunisia and to assess the efficiency of AI for Magrebin ovine breeds, the results related to different semen conditioning techniques and different AI procedures. The main factors affecting AI results are also presented. Finally, this chapter presents different strategies to improve AI efficiency at farm level in the future and the challenges to extrapolate experimental AI techniques to field conditions at a large scale.

**Keywords:** semen preservation, cervical insemination, laparoscopy, sheep

#### **1. Introduction**

Sheep farming is of great economic, social and environmental interest in all countries with a Mediterranean climate [1]. It remains an important activity in the southern Mediterranean countries particularly in Morocco, Algeria and Tunisia considering its adaptation to the majority of the countries' agro-ecosystems, which is due to the hardiness of the dominant breeds and to the flexibility of production systems in relation to socio-economic and land contexts [2–8]. Yet, breeding techniques at farm level are till nowadays basic and traditional based on pasture, which is subjected to the current issue of global warming causing severe rangeland degradation [5, 9]. This leads to substantial limited productive capacity of sheep characterized by a low annual productivity rate ranging between 0.66 and 1.24 lambs for most breeds in the Maghreb region [3, 4, 7] added to a relatively low carcass weight (about 15 kg) [10, 11] and low survival rate [7] causing a lack of red meat production.

In this framework, artificial insemination (AI) offers a powerful tool to develop ovine sector through speeding up selection programs and spreading the genetic progress. Thus, improving flocks productivity became a national objective in the Maghreb countries where several enhancing breeding programs have been initiated in order to remedy these problems starting with improving the control of

reproduction and creating national centers for ovine using AI. Ovine AI offers enormous opportunities to support the sector development, by accelerating the programs of selection and dissemination of genetic progress. AI allows outof-season reproduction and therefore milk or lamb productions that are better distributed throughout the year in response to the needs of the market. In addition, AI allows breeders to have access to the best mated male for herd renewal while limiting health risks. Furthermore, AI permits the multiplication of genotypes and limiting applied consanguinity, without multiplying the number of progenitor in the herd [12].

In recent years, continued improvements of this method in bovine, caprine and poultry coupled with a growing demand for the application of AI at farm level, as the numerous benefits it offers, are being increasingly recognized. Yet, ovine AI has progressed rather slowly in terms of breed improvement in comparison with the aforementioned species [13, 14]. The earliest documentation on a large scale ovine AI has been reported in Russia by Miovanov in 1938 [13] than spread to China and central Europe. It was not until the 80′s that the first reports of sheep cervical AI that was documented in the Maghreb region by Khaldi and Farid in 1981 in Tunisia [15] and Manar in 1987 in Morocco [16]. Later in 1992, that was the first documented laparoscopic insemination of French breed ewes in Morocco [17]. Ever since, ovine IA has gained researchers′ interest and several works have been developed since in order to investigate factors influencing IA results in Maghrebin sheep breeds and to improve fertility rate of inseminated ewes.

According to this context, our work aims to highlight the actual situation of assisted reproduction in sheep with a special emphasis on rams′ semen collection and preservation and AI in the Maghreb region. We will first address the importance of ovine farming and its limits in this region. Then, we will review the current status of AI in sheep particularly at farm level. Finally, we will focus on future consideration to enhance assisted reproduction and to discuss how to evaluate the applicability of ovine AI at farm level in the Maghreb region. Electronic databases (Elsevier, PubMed, and Web of Science) along with some official reports and thesis documents were consulted for an appropriate literature review. A total of 55 suitable references were considered for this chapter from 1981 till now.

#### **2. Overview of sheep farming in the Maghreb region**

Sheep farming in the Maghreb region is well developed mainly due to its flexibility and hardiness of the dominant breeds as well as spatial complementarities to agricultural production [2–8, 18]. It closely depends on the climatic conditions of the year as it is based on traditional farming systems related to pasture availability.

#### **2.1 Place of sheep breeding in the Maghreb countries**

For the whole Maghreb region, the total number of sheep increased from 23 million during the 1960s to nearly 30 million in 1970s and reached 34.9 million in 1980s to stabilize around 37 million during the 1990s. The most important increase in ovine flocks has been registered from 2005 to 2019. The total number of sheep in the Maghreb region reached more than 57 million by 2019 [19]. This rapid increase in the herd, the strongest in Algeria followed by Morocco (**Figure 1**), seems to have been favored by the short-term public efforts during the droughts and the distributions of barley and fodder to resist the disastrous damages of drought and climate change on pasture [20]. In Tunisia, the evolution of the herd was fluctuating before the sixties indicating a close dependence of sheep farming on climatic conditions.

*Ovine Artificial Insemination in the Maghreb Region: Present Status and Future Prospects DOI: http://dx.doi.org/10.5772/intechopen.100273*

**Figure 1.** *Evolution of sheep stocks in the Maghreb countries (million heads).*

Then through public safeguard campaigns and the subsidy for concentrated feeds, the sheep flocks have stabilized since the late eighties around 6 million head [4, 5]. The main objective of the public authorities was to increase sheep stocks and consequently to increase the production of meat for which these countries are in deficit.

Sheep farming is mainly intended for meat production in the Maghreb region, and most breeds were not selected for dairy traits, except for Sicilo-Sarde in Tunisia [21, 22]. It contributes significantly to the value of agricultural production in all Maghreb countries and insures a major part of self-sufficiency in meat consumption. For instance, ovine meat contributed with up to 48% in the total production of red meats in Tunisia estimated at 120,000 tons [4]. In Morocco, ovine stock ensures a meat production of around 130,000 tons of carcass equivalent yearly [23]. Likewise, according to official statistics of the ministry of Agriculture and Rural Development in 2017, Algeria has 26 million head of sheep and produces 325,000 tons of sheep meat, which ranked the country 5th in the world in terms of sheep meat production [8].

#### **2.2 Characteristics of the main ovine breeds in the Maghreb region**

The Maghreb region is characterized by different climatic zones from the Mediterranean coast to the oases of the Sahara. These diversified ecological conditions and climate types offered an extraordinary diversity of indigenous sheep breeds well adapted to their respective environments [24]. Except for Sicilo-Sarde breed in northern Tunisia, all sheep breeds in the Maghreb region were selected for meat and wool remains the next desirable product. Due to their proximity and commune history of the region's people, genetic flow between all Maghrebin breeds has shaped a rich livestock heritage [24, 25]. The Maghrebin sheep stock consists of several indigenous breeds with particular transboundary breeds (i.e., Algerian Hamra also called Beni-Guil in Morocco, D'Man and Ouled-Djellal). In this section, we present the main Maghrebin breeds that have been reported in the literature to receive genetic breeding programs and particularly artificially inseminated (**Table 1**).

The Algerian sheep population consists of nine breeds (Ouled-Djellal, D'man, Hamra, Rembi, Taâdmit, Sidaoun, Tazegzawt, Berbere and Barbarine), strongly adapted to harsh environmental conditions [26]. Some other non-official breeds were also reported originally introduced from Morocco and sub-Saharan Africa like Moroccan Srandi ou Sardi and Ifilene [27]. Due to the increasing farmers'

*Ovine Artificial Insemination in the Maghreb Region: Present Status and Future Prospects DOI: http://dx.doi.org/10.5772/intechopen.100273*

**Table 1.** *Main sheep breeds in the Maghreb region.*

preference to a single breed, Ouled-Djellal currently accounts for more than 63% of the Algerian sheep population. According to Mason [28], Moroccan sheep population is composed of some twenty different breeds well adapted to their variant ecosystems and tolerant to harsh climates. Currently, the most important breeds are Sardi, Timahdite, Béni Guil (also called Hamra in Algeria), D'man and Boujaâd. These breeds were phenotypically characterized and their breed standards were established since the beginning of 1980s [29].  The main exogenous sheep breed found in Tunisia is the Moroccan prolific D′man breed, which represents about 0.25% of the total sheep population in Tunisia [30].

Despite the great genetic diversity, sheep productivity remains insufficient in the Maghreb countries. As a whole, it would be only 12 kg of lamb at weaning per ewe per year with 0.66 to 1.24 lamb/ewe/year in Morocco [3] and about 12.8 kg of lamb per ewe per year with 1.13 lamb/ewe/year in Algeria [7]. Similarly, the productivity of most Tunisian breeds was estimated about 0.8 weaned lambs per ewe per year and about 14 kg of lamb at weaning per ewe per year [4, 31] because of the low performance of the ewes and lambs. Low fertility, prolificacy, and high neonatal mortality are reported for most local breeds under extensive management system coupled with insufficient mastery of breeding techniques in terms of genetics, feeding and reproduction. Along with improving management's techniques and feeding conditions, researchers have recommended adoption of reproductive biotechnologies to improve the performance of these indigenous breeds and disseminate genetic progress [32]. Thus, several studies have been conducted to meet this need. In the following part of this chapter, we will review the most relevant works carried out related to reproductive biotechnology and AI in Maghreb ovine breeds with a particular emphasis on most important results at farm level.

#### **3. Current state of ovine AI in the Maghreb region**

The first documented studies on AI of ovine species in the Maghreb countries were reported during the 1980′s following the establishment of artificial insemination centers by public authorities. Since its creation in 1975, the sheep breeding program in Tunisia has been managed by the Office of Livestock and Pasture (OEP). This program aimed to allow the dissemination of improved genes acquired by the herds controlled in sheep farms in different regions [33]. Sheep semen collection, control and conditioning are provided by the services of the Genetic Improvement Direction (DAG) of the OEP-trained pure local breed rams of the center (**Figure 2**). In Morocco, two artificial insemination centers (Fouarat and Aïn Jemaa) exist providing ovine AI services. Since the 90s, the Ministry of Agriculture has set up a laboratory for the semen storage of small ruminants at Aïn Jemaa Center. The goals assigned to this center were to produce and preserve ram semen deriving from five local breeds and several imported breeds (Ile de France, Merino and Lacaune) and the assessment of fertility of frozen semen [16]. It was only later (2006–2011) that the creation of three regional Centers of Ovine Artificial Insemination (COAI) in Algeria has led to

**Figure 2.** *Trained rams for semen collection a: Fat-tailed Barbarine b: Noire de Thibar (DAG, Sidi Thabet, Tunisia).*

#### *Ovine Artificial Insemination in the Maghreb Region: Present Status and Future Prospects DOI: http://dx.doi.org/10.5772/intechopen.100273*

the introduction of this technique in sheep and its diffusion lately at national level [34]. Even though the creation of these centers had enhanced research on AI of local ovine breeds, the use of AI at farm level is till nowadays very limited and applied on few thousands ewes per year with little success.

#### **3.1 Semen collection and preservation**

One of the most limiting factors of the large scale use of AI in ovine selection programs is the difficulty of ram′s semen preservation and cryoscopy. Thus, the use of fresh semen in trans-cervical insemination is the most common practice. For this reason, numerous studies have been recently performed with the goal of optimizing sperm cryopreservation protocols in this specie [35, 36]. However, there is paucity in studies about sperm collection procedure in Maghrebin local breeds. Semen collection from large numbers of untrained rams makes AI with fresh sperm at farm level difficult to perform.  Semen can be collected from live animals by artificial vagina (AV) or electrical stimulation (EE) [35]. Semen collection with an AV simulates natural conditions, but usually requires a preliminary training period of rams [35], whereas obtaining semen from a large number of rams, EE could be a useful and faster procedure [13]. However, most field trials conducted in Maghreb region were only performed with AV semen collection techniques (**Figure 3**) after 2-week period of rams training to ejaculate in AV [36, 37].

After collection, sperm must be diluted and cooled slowly but progressively from collection temperature (+32°C) to storage temperature (+15°C or + 5°C) in order to slow down the basal metabolism of spermatozoa from ejaculation until AI. Different extenders were used during liquid and frozen storage to improve sperm motility, viability and functional integrity of ram sperm membranes and ultimately success rate of consequent AI. In a previous work [32], different extenders were tested during liquid (15°C) and cryoscopy conservation of ram sperm. The use of

**Figure 3.** *Semen collection with an artificial vagina in the Tunisian " Noire de Thibar " ewe.*

skimmed milk with sulfamid and antibiotics during liquid storage gave satisfactory results as sperm motility score ranged between 3.1 and 4.0 and sperm viability ranged between 52 and 71% during the mating season. These authors also tested the effect of two extenders (skimmed milk with egg yolk *vs.* Tris with egg yolk, citric acid and fructose) during frozen conservation. They used glycerol as cryoprotectant and antibiotics for both extenders. The freezing procedure was evaluated. Automated cryoscopy led to satisfactory post-thawed sperm quality with both extenders (over 3.1 motility score and 44% viable spermatozoa) recommended for intra-uterine insemination. However, manual cryoscopy caused severe damages of the spermatozoa and gave very low sperm motility and viability. Recent study [38] performed using four extenders: two based on egg yolk (egg yolk Tris and Tryladil), one based on milk (skimmed milk or Colas extender (use in equilibration and freezing)) and one to soy lecithin (Andromed) showed that the skimmed milk presented most advantageous in the preservation of the semen at 5° C. While diluents containing egg yolk have best preserved semen quality of ram INRA180 breed during the freezing procedure, it was found that ram effect was a significant factor in sperm storage in this study and it was found that sperm from ram number 2 showed the better resistance to storage, either in liquid or in frozen state [38].

To reduce the oxidative stress during storage process, several extenders and protective components have been tested with a particular emphasis on locally produced antioxidant agents in some plant extracts. Recently, in [39–41] it has been shown that the addition of argan oil and cactus seed oil with small amounts to Tris egg yolk/skim milk extenders increased the total sperm motility, progressive motility, viability and membrane integrity, and decreased the spontaneous and induced lipid peroxidation and DNA fragmentation in ram semen at 15 and 5°C temperatures. Similar effects were reported when 1% of *Opuntia ficus-indica* extract was added to extenders such as Tris or milk during liquid storage up to 72 h of storage [41].

#### **3.2 Estrus synchronization and insemination timing**

In sheep, most AIs are practiced with fresh semen on induced estrus within a few hours after the semen collection (optimum: 5 hours-maximum: 10 hours). AI efficiency is closely related to estrus induction and synchronization procedure.

Several synchronization techniques were tested for local breeds in the Maghreb region over the years. The most commune estrus synchronization procedure is based on the use of vaginal devices (sponges) impregnated with 30–40 mg of fluorogestone acetate progesterone implants for 14 days and equine Chorionic Gonadotropin (eCG) intra-muscular injection on removal day (**Figures 4** and **5**) [32, 34, 38, 42, 43].

Two synchronization treatments were tested for different Moroccan sheep breeds [44] using progesterone implant coupled with eCG or prostaglandin analog injection. Lambing rate of D'man ewes was 34.9 and 21.7% (respectively for ewes treated with PGF2α and progesterone), while this rate was 39.1 and 13%

**Figure 4.** *Simplified estrus synchronization protocol for ewes.*

*Ovine Artificial Insemination in the Maghreb Region: Present Status and Future Prospects DOI: http://dx.doi.org/10.5772/intechopen.100273*

**Figure 5.** *Intravaginal sponge insertion and PMSG injection in the Tunisian Barbarine ewe.*

respectively for ewes treated with PGF2α and progesterone for Timahdite breed. A second trial focused on eCG dose (250 IU *vs.* 500 IU) in D'man and Sardi ewes, and the results showed a very low fertility after AI, from 10 to 40 in D'man and 12 to 28.5 in Sardi respectively for 250 and 500 IU.

Recently, the effect of eCG doses on fertility parameters' of Moroccan Boujaâd ewes were tested [45] and the use of 300 IU of eCG after 14 days of progesterone vaginal sponge insertion was recommended. With 400 IU, ewes had significantly higher prolificacy associated with higher neonatal death. On the other hand, eCG treatment has been associated with lower fertility rates on ewe′s consecutive cycles. The use of ram effect coupled with progesterone treatment instead of eCG injection in Barbarine ewes was also tested [43]. Results showed that substituting eCG treatment by the ram effect as synchronization treatment prior to artificial insemination could lead to satisfactory lambing rates.

Most reviewed works recommended performing cervical AI after 55 ± 1 hour after progesterone sponge removal using fresh or cooled semen [32, 34, 38, 42, 45] giving satisfactory results going up to 100% of synchronized ewes and pregnancy rate varying between 52 and 88%. Earlier studies tested performing AI after 60 h of progesterone implant removal in D′Man and Timahdite breeds. At this timing, the fertility rate was low for both tested breeds with 21.7% and 13% respectively for D′Man and Timahdite ewes. Moreover, it has been shown that cervical insemination of Barbarine ewes after 55, 56 and 57 h after hormonal treatment and progesterone sponge removal gave satisfactory results with respectively 59, 48.6 and 52% fertility rates [42]. However, when AI was delayed to 58 and 60 h after hormonal treatment, fertility rate dropped to 25 and 26.5%, respectively. Thus, it was recommended that large flocks should be divided into groups of 25 ewes in order to prevent time shifts during the procedure and prolonged the interval between hormonal treatment and AI application.

#### **3.3 Advances in ovine AI in the Maghreb region**

In the Maghreb region, ovine AI is till nowadays limited to experimental centers, and AI at farm level is not yet extended. In Tunisia, few thousand ewes are yearly inseminated as part of DGA activities to limit the effect of consanguinity in small pure breed herds, particularly Sicilo-Sarde and Noire de Thibar [46]. Thus, most of the presented data in this review remain experimental. Using fresh or cooled ram

**Figure 6.** *Cervical AI in the Tunisian "Noire de Thibar" ewe.*

semen, intra-cervical (**Figure 6**) deposition of semen techniques results in acceptable fertility rates. However, using frozen-thawed semen, at present only intrauterine insemination gives acceptable pregnancy rates that remain difficult to achieve and to disseminate at large scale because of its cost and complexity.

#### *3.3.1 Cervical insemination*

This section summarizes the main findings in cervical insemination in the Maghreb ovine breeds. The conception rate to AI is calculated as the proportion of pregnant ewes detected by ultrasound scan at early stage of pregnancy or lambing ewes from inseminated ones. Results varied widely depending on the conditions and experiment protocol. In many studies, the ewe′s breed has been found to have a strong effect on the pregnancy rate after AI. In an industrial breed crossing trial, the Sardi, Timahdite and Boujaâd Moroccan local ewes were crossed with rams of Ile de France, Merinos and Lacaune [47]. Cervical insemination was performed at 56 h after the sponge removal with fresh cooled (15°C) semen of 1.6 × 109 spz/ml concentration. Results showed the superiority of Sardi breed crossed with Merinos compared to the others with fertility rate 90.48% and prolificacy of 136%, while fertility rate ranged between 42.31 and 76% for Timahdit and Boujaâd breeds crossed with Ile de France, Merinos and Lacaune rams. The male may greatly influence fertility results after cervical AI. Variation in fertility of ram ejaculates exists independently of the sperm quality and after cervical inseminations with fresh semen [47].

A significant ram′s age effect on semen quality in Algerian Ouled Djellal breed was reported [48]. The concentration and the mass motility were significantly higher in adult males. As expected, adult rams had significantly higher fertility and prolificacy (74.88 and 161.87%, respectively) compared to young rams (50.08 and 120.91%, respectively). However, the age of the female did not affect their fertility. They also reported that season had a highly significant influence on the ejaculate volume but not on the concentration and mass motility of spermatozoa. In a Tunisian study [38], fertility rate of the Barbarine ewes ranged between 32 and *Ovine Artificial Insemination in the Maghreb Region: Present Status and Future Prospects DOI: http://dx.doi.org/10.5772/intechopen.100273*

41% with a significant effect of animal management was reported. Similarly, fertility rates ranged between 38 and 73% and prolificacy from 1.03 to 1.24 in the same breed with a great flock effect indicating the importance of body condition and nutritional background of animals to improve success rate of AI have been reported [43].

A study on AI at farm level carried out during spring mating following induced estrus of the four main indigenous Tunisian ovine breeds: Sicilo-Sarde (SS), Noire de Thibar (NT), Queue Fine de l'Ouest (QFO) and black head (BTN) and red head (BTR) Barbarine [46] reviled that the fertility of ewes inseminated out-of-season varied from 32 to 74% with an average of 47.56 ± 9.94%. The use of chilled semen (5°C) significantly (p < 0.01) reduced the success rate of AI with 43.76 ± 7.56% versus 55.95% ± 9.56% using the fresh semen. The superiority of the SS breed over the Tunisian meat breeds was perceived. An important effect of breeding management was detected, showing the importance of preparing ewes before using AI [46]. In another field trial aiming to improve the productivity of Timahdite breed, the fertility results obtained were as high as 60% of lambing rate, while in a field study carried out on herds from the same region (Middle Atlas), the fertility rates were 60, 44 and 41.5% respectively at Irklaouen, Timahdite and Ain Leuh communes [16]. The great variation of AI results at experimental level and at farm level indicates the potential of this biotechnology on large scale and the possibility of disseminating this technique. However, much still to be done in order to get reproducible results and limits the effect of factors of variation.

#### *3.3.2 Laparoscopic insemination*

Laparoscopic intrauterine insemination (LAI) is up-to-date the only technique that guarantees adequate fertility with frozen-thawed semen. Although it is still performed on experimental level, it is particularly interesting choice to use sperm with high genetic value [32, 49] and/or when semen is imported [17, 32, 49] or postthawing semen quality is poor [50]. At field level, several factors limit the diffusion of LAI starting with its complexity and the need for highly trained technicians and advanced equipment to perform it. This procedure is also very expensive and have other problems related to animal welfare [51]. LAI offers higher and more constant fertility rates than cervical AI. In the Maghreb region, a few LAI trials have been performed in order to improve genetic value of some local breed, particularly small group breeds with high consanguinity risk.

It has been reported the use of intrauterine inseminations between 2005 and 2007 with Sarde-frozen semen imported from Italy in order to overcome the scarcity of sires and to alleviate consanguinity hazards on the Sicilo-Sarde dairy breed in Tunisia [49]. A renewal of phenotypic variability was observed mainly at the level of the herds, which benefited the most from this crossing. These animals, of different genetic types, took advantage of the superiority of the Sardinian breed, which improved several criteria of production or conformation. Results of gene injection protocol showed that fertility, prolificacy and mortality rates ranged from 53 to 68%, 157 to 184% and from 5 to 11%, respectively. A similar experiment was conducted in Morocco to improve the genetic value of imported French breeds used for commercial industrial crossbreeding since the nineteenth century. The genetic value of these breeds has been compromised by high consanguinity, since the import of live animals is restricted, frozen semen was the ultimate alternative inseminated with laparoscopic technique. Results ranged between 38.5 and 75.5% for fertility and 126.7 and 168.2% of prolificacy depending on breed with the highest results registered in the Solognot breed [17]. The gestation rate of Noire de Thibar ewes inseminated with frozen-thawed semen of Brune Noire Suisse rams with LAI ranged between 52 and 81% [32].

Even though, LAI is still very limited in the Maghreb countries, it is considered a good way to enhance genetic value of rams particularly in insemination centers and could be a powerful tool to increase genetic selection pressure.

#### **4. Future prospect in ovine AI**

In the Maghreb region, ovine breeding is developing continuously particularly in Algeria and Morocco since small ruminants are better suited to their production systems and to their harsh climate than cattle. Thus, it is likely that there will be an upsurge in the use of AI in these species in the future, with an emphasis on improving production traits by the injection of superior genes. Introducing innovative solutions is increasingly adopted by livestock holders including acceptance of reproduction biotechnologies [52]. This would allow a large-scale diffusion of AI in the future. However, one of the greatest challenges against any genetic improvement program at national level *via* AI is the improvement of animal husbandry and management to ensure the success of such program. AI would offer little help in areas where basic husbandry skills are inadequate.

The creation of breeding centers for pregnant young ewes and rams is essential to meet the needs of sheep breeders and contribute to the genetic improvement of the herd particularly in the dairy Sicilo-Sarde breed [53], Noire de Thibar and most breeds with relatively reduced numbers. In Morocco, for the first time, a private AI center has been founded with an ambitious program but it is still not functioning as expected [16]. Currently, this center is focusing on many goat projects and capacity building reinforcement of sector stakeholder, while little is done or has to be done in sheep. Future projects will involve insemination of 2000 ewes in Boujaâd breed and should later be extended to Sardi breed.

#### **4.1 The cost of artificial insemination**

One of the most important challenges against AI diffusion at large scale in the Maghreb region is its cost. Along with improving fertility rate following AI, researcher should focus on reducing intervention cost. For instance, AI charge in bovine specie in Tunisia is estimated around 3 € for the first IA, 2.45 € for the second and 1.84 € for the third and over till successful insemination for the same cow using locally produced pure breed semen. At this cost, coupled with the value of the animal, bovine AI is very valued and applied in both dairy and meat cattle. Yet the cost is significantly higher when the breeder chooses the use of imported semen and/or sexed semen to inseminate his cows, the cost is usually accepted given the importance of the genetic value of the expected offspring. On the contrary, the ovine AI real cost remains higher with lower commercial value of the product. Coupled with the estrus synchronization treatment, AI of ewes would be very expensive. This leads to the farmers' refusal to practice this approach. The use of more natural technique such as ram effect could reduce direct charges of the technique, although natural techniques lead always to very variable results. Even though the ovine IA services provided by governmental centers of AI are until nowadays free of charges in Maghreb countries, they are still localized and offer very limited services since the use of frozen semen is yet to develop. Information about the real cost of ovine AI is not available in the Maghreb region and the study of the economic impact of this biotechnology is strongly recommended.

*Ovine Artificial Insemination in the Maghreb Region: Present Status and Future Prospects DOI: http://dx.doi.org/10.5772/intechopen.100273*

#### **4.2 Sperm cryopreservation**

Most studies presented in this review had a particular interest to the storage conditions at 15°C and 5°C to prolong the storage time to 8–24 h. The extended refrigeration period would reduce the dependence of the AI centers. However, the needed doses were high (around 109 spz/ml) [47, 48]; thus, the number of doses/ ejaculate remains very low. The effect of cryconservation [37] and different cryprotectors with different extenders in liquid preservation have been studied succinctly [40–43, 45], and most studies have been performed *in vitro* conditions. Most results showed good sperm preservation in liquid up to 24 h yet further research is required to design a valid strategy for the preservation of liquid semen from rams in the medium term 48 to 72 h. The effect of cryopreservation and freezing on ovine sperm was evaluated. Three steps were tested including storage in the liquid state (5°C), equilibration with four different extenders (Tris-egg yolk, Colas (skimmed milk based extender), Tryladil and AndroMed®) and then freezing [37]. The results showed that the time of storage at 5° C, equilibration and freezing have negatively affected the sperm quality. The skimmed milk had the best results at 5°C for 48 h compared to other extenders, while Tris-egg yolk extender was the best to preserve semen quality of rams during the freezing procedure with 72% total spermatozoa motility. These experiments revealed promising results *in vitro* but further studies will be needed to evaluate the effect of cryopreservation on sperm′s fertility.

#### **4.3 Challenge of sperm deposit site: transcervical intrauterine insemination, the technique to develop**

The development of a non-surgical AI procedure that could be performed efficiently and repeatedly with constant results will have tremendous implication on selection programs and genetic progress of sheep. Deposit site of semen during AI in the ewe′s genital tract has direct repercussion on fertility rate. Cervical AI leads to relatively low fertility rates even when using fresh semen. The particular, highly complex structural arrangement of ewe's cervix prevents easy transcervical passage and intrauterine deposition using conventional AI catheters [54]. Since the 1970, efforts have been made in order to access the uterine lumen by the transcervical route, which would allow the use of frozen-thawed sperm [55]. The success of transcervical intrauterine insemination depends on several factors, including cervical dilatation, the design of the catheters and the used procedure. Several works had investigated all these aspects, but none of these have been tested on local Maghreb breeds. A lot have to be done in order to promote AI of the ovine specie around the world and particularly in the Maghreb region.

#### **5. Conclusion**

In summary, despite the importance and the continuous progress of the ovine sector in the Maghreb region, particularly in Algeria and Morocco, AI is not yet an operative tool for the ovine breeding development and selection. Fertility rate following AI is very fluctuant and remains low 30 to 76% in most cited literature depending on the breed, the technique and the use of fresh, chilled or frozen semen. The development of AI of sheep became crucial to enhance genetic progress of this specie and to preserve some indigenous breeds with particular rustic characteristics and well adapted to their harsh environment. Many of the published studies are conducted under experimental conditions with a low number of animals; thus, it is difficult to extrapolate their conclusions to field conditions at a large scale. The use of frozen-thawed semen along with a non-surgical technique, if mastered, could enhance and promote the use of AI at farm level and accelerate the genetic progress of ovine. The combined protocols (modified catheters plus dilator substances) could be the beginning of the solution for transcervical insemination, but the complexity of the technique, the time spent in cervical penetration and the side effects that it produces are key factors for the success and dissemination of the TCAI and should be optimized to achieve an efficient procedure.

## **Conflict of interest**

The author declares no conflict of interest whatsoever.

## **Author details**

Moufida Atigui1 \* and Mohamed Chniter2

1 Higher School of Agriculture Mateur, Improvement and Integrated Development of Animal Productivity and Food Resources, University of Carthage, Mateur, Tunisia

2 Department of Animal Sciences, National Institute of Agronomy of Tunisia, University of Carthage, Tunis, Tunisia

\*Address all correspondence to<EMAIL_ADDRESS>
© 2021 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.

*Ovine Artificial Insemination in the Maghreb Region: Present Status and Future Prospects DOI: http://dx.doi.org/10.5772/intechopen.100273*

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#### **Chapter 7**