**1. Introduction**

**Abstract:** We investigated

For decades, soybean meal has been utilized globally in poultry diets, with maize providing the primary source of dietary energy [1]. Yet these feed ingredients are often inaccessible to developing countries for poultry production due to high cost and availability of soybean and corn, with the United States, Brazil, and Argentina being the largest producers and exporters of soybeans [1]. Hence, other protein sources like canola meal, peanut meal, fishmeal, and blood meal are also utilized.

In India, Ghana, and Nigeria, peanut meal is commonly utilized as a protein-rich poultry feed ingredient [2–6], yet in the US, 85% of peanut production is for peanut butter and snacks, with the remaining 15% crushed for oil [7]. Early poultry feeding trials established that peanut meal prepared from conventional normal-oleic peanuts (22–30% protein and 44–56% total fat, with a total fatty acid profile of 52% oleic acid and 27% linoleic acid) is a reasonable poultry feed ingredient [8,9]. Additionally, limited studies have investigated the use of high-oleic peanuts (22–30% protein and 44–56% total fat) with an 80% oleic fatty acid and 2% linoleic fatty acid profile as an alternative poultry ration. Toomer et al. [10] reported that eggs produced from Leghorns in peak egg production (40 weeks of lay) fed high-oleic peanuts had increased β-carotene and oleic fatty acid content, with increased yolk color compared to conventional eggs, with no significant differences in hen performance (with the exception of egg mass) or egg quality between

treatment groups. However, egg weights produced from hens in peak egg production (40 weeks of lay) fed a high-oleic peanut diet were significantly smaller in mass relative to conventional eggs at all time points measured [10]. Hence, the primary aim of this project was to determine the effect of feeding a high-oleic peanut or an oleic acid diet on the size/mass of the eggs produced by older production hens (57 weeks of lay). The establishment of a feeding regimen to reduce the production of oversized eggs commonly seen with increasing production age is of great commercial interest [11]. Based on our previous findings [10], we conjecture that eggs produced from hens fed a high-oleic peanut or oleic acid diet will have enhanced β-carotene and unsaturated fatty acid content, with smaller egg size (mass), as compared to eggs produced from hens fed a conventional diet. Moreover, we aimed to investigate the effects of a high-oleic peanut diet on the sensory attributes of the eggs produced.

## **2. Materials and Methods**

All animal research procedures used in these feeding trials were approved by the North Carolina State University Institutional Animal Care and Use Committee (IACUC #17-001-A).

#### *2.1. Experimental Design, Animal Husbandry, Dietary Treatments, and Hen Performance*

All experimental diets were formulated in Concept 4 (level 2, version 10.0) software to be isonitrogenous (18% protein) and isocaloric (3080 kcal/kg metabolizable energy), with an estimated particle size between 800 and 1000 μm (Table 1). The control diet (Treatment 1) was prepared as a conventional layer diet with defatted soybean meal and corn, while Treatment 2 was prepared using aflatoxin-free whole non-roasted unblanched high-oleic peanuts. Peanuts were crushed using a Roller Mill to form crumbles, prior to inclusion in Treatment 2. Treatment 3 was prepared by supplementing the control diet with 2.64% (% by weight) of food-grade oleic fatty acid oil (Millipore Sigma, Burlington, MA, USA). Each of the experimental diets were supplemented with vitamin, mineral, and selenium premixes manufactured at the NC State University Feed Mill (Raleigh, NC, USA) to meet and/or exceed poultry requirements for vitamins, minerals, and selenium. All experimental diets were analyzed by the North Carolina Department of Agriculture and Consumer Services and the Food and Drug Protection Division Laboratory (Raleigh, NC, USA) for aflatoxin and microbiological contaminants.

Brown Leghorn hens were selected for use in this study from the University Flock, NC State University (Raleigh, NC, USA). In total, 99 Brown Leghorn hens (57 week of lay) were assigned to three dietary treatment groups for 8 weeks: (1) Conventional diet; (2) HOPN diet; (3) OA diet. There were three replicates per treatment, with hens individually housed in battery cages (each cage measured 12 inches wide × 18 inches deep × 18 inches height) in one room at the Chicken Education Unit, NC State University (Raleigh, NC, USA). Hens were provided feed and water ad libitum and 14 L:D for 8 weeks. Body weights were recorded for each individual hen at week 1 and week 8, with feed weights recorded weekly. Shell eggs were collected, enumerated, and weighed daily. Total number of eggs produced per replicate and per treatment was calculated for each experimental week and for the total 8 week feeding trial. The average feed conversion ratio (FCR) was calculated as total feed consumed over the 8-week feeding (kg)/dozens of eggs produced for each treatment group over the 8-week feeding trial.

#### *2.2. Egg Quality and Grading*

Bi-weekly (0-week, 2-week, 4-week, 6-week, and 8-week), 36 eggs were randomly selected with 12 shell eggs per treatment (4 eggs randomly selected from each replicate) for quality assessment and USDA grading. Fresh shell eggs were collected on the day of quality assessment and USDA grading. Shell eggs were analyzed for DSM yolk color score, vitelline membrane strength, Haugh unit, and shell strength by the Laying Hen and Small Flock Management Lab, Prestage Department Poultry Science, NC State University. Haugh unit values were determined using methods described by Haugh [12] and were recorded with the Technical Services and Supplies (TSS) QCD system (Dunnington, York, United Kingdom). The QCD system was calibrated to the DSM Color Fan, consisting of a series of 15 colored plastic tabs with a range of yolk colors from light yellow to orange red (color index 1 to 15), defined by Vuillemier [13]. In general, a texture analyzer (TA.XTplus) was used to measure the shell strength and vitelline membrane strength by the breaking strength using a 5-kg load cell per the manufacturer's instructions (Stable Micro Systems, Surrey, United Kingdom), with measurements in grams of force. Vitelline membrane strength was determined using methods described by Jones et al. (2005), with a 2 mm/second test speed and 0.0001 kg trigger force [14]. Modified methods of Jones et al. (2002) were used to measure shell strength with a 2 mm/second test speed and a 0.001 kg trigger force [15].

1 Three isocaloric, isonitrogenous (18% protein) formulated diets were fed to Brown Leghorn (57 week of lay) hens for 8 weeks. 2 Treatments: control = conventional soybean meal and corn mash diet, HOPN = unblanched high-oleic peanut crumbles (20%) and corn mash diet, OA = control diet supplemented with 2.64% food-grade oleic fatty acid oil. 3 High-Oleic Peanuts = unblanched raw whole high-oleic peanut crumbles. 4 MYC-Out™ = mycotoxin binder and feed antioxidant manufactured Adisseo (Alpharetta, GA, USA). 5 Mineral premix, manufactured by NCSU FeedMill, supplied the following per kg of diet: manganese, 120 mg; zinc, 120 mg; iron, 80 mg; copper, 10 mg; iodine, 2.5 mg; and cobalt. 6 Vitamin premix, manufactured by NCSU FeedMill, supplied the following per kg of diet: 13,200 IU vitamin A, 4000 IU vitamin D3, 33 IU vitamin E, 0.02 mg vitamin B12, 0.13 mg biotin, 2 mg menadione (K3), 2 mg thiamine, 6.6 mg riboflavin, 11 mg d-pantothenic acid, 4 mg vitamin B6, 55 mg niacin, and 1.1 mg folic acid. 7 Selenium premix, manufactured by NCSU FeedMill, = 1 mg Selenium premix provided 0.2 mg Se (as Na2SeO3) per kg of diet.

#### *2.3. β-Carotene, Lipid Content, and Fatty Acid Analysis*

All experimental diets were analyzed for lipid content, fatty acid, and β-carotene content in triplicate by an external vendor ATC Scientific (Little Rock, AR, USA), using AOAC approved methods. Gross energy analysis of feed samples was performed by ATC Scientific using an adiabatic oxygen bomb calorimeter with standard methods. Biweekly, a total of 45 eggs were randomly selected, with 15 eggs per treatment (5 eggs per replicate) for lipid content, β-carotene, and fatty acid analysis by ATC Scientific using AOAC approved methods. Each egg sample was mixed for homogeneity in a whirl-pak® (Millipore Sigma, St. Louis, MO, USA) bag for 3 min using a Smasher™ Lab Blender (Weber Scientific, Hamilton, NJ, USA). Subsequently, all egg samples were frozen at −20 ◦C and

stored frozen until chemical analysis within 2 weeks of collection. Frozen homogenous egg samples were shipped on dry ice overnight to vendor for analysis within 2 weeks of collection. Lipid (total cholesterol, crude fat) and fatty acid analysis of homogenous egg samples and feed samples were analyzed using direct methylation methods, as described by Toomer et al. [10]. Total cholesterol was measured as mg cholesterol/100 g sample weight (feed or egg), while crude fat was measured as a percentage of gram crude fat/gram sample weight (feed or egg). Fatty acid content was measured as a percentage of gram of fatty acid/gram total lipid content of a sample (feed or egg). Methods used to determine β-carotene content in eggs are detailed in the AOAC 958.05 [16] color of egg yolk method.  Cooking Methods and Consumer Acceptance Testing of Scrambled Eggs*

The Sensory Service Center, in the Food, Bioprocessing, and Nutrition Sciences Department, NC State University (Raleigh, NC, USA), performed all sensory testing and data analysis of egg samples. The sensory protocol was reviewed and deemed exempt by the NC State University Institutional Review Board for human subjects. Scrambled egg samples were prepared following safe food handling practices. During preparation and service, all team members wore gloves, hairnets/hats, and lab coats. Whole shell eggs were received and refrigerated at 4 ◦C upon arrival. On the day of testing, preparation of raw eggs was completed on a separate table from the cooking/serving areas of scrambled eggs to prevent cross contamination of any microbial hazards, and gloves were changed and hands were frequently washed during any transition from raw to cooked product.

On the day of the sensory evaluation, approximately 150 shell eggs per treatment were cooked. In total, 10 sets of 15 eggs/treatment were cracked into a bowl and beat together until homogenous. A large non-stick pan was heated over medium heat for approximately 1 min. The homogenous egg mixture was added to the heated pan and stirred slowly with a wooden spatula, bringing in the mixture from the edges of the pan for 3.5 min and subsequently removing the eggs from the pan. Scrambled egg samples from each of the three treatments were placed in labeled aluminum pans, covered with aluminum foil, and held in a heated holding cabinet at 180 ◦F to maintain quality. Scrambled egg portions were dispensed into lidded soufflé cups with three-digit codes to identify treatment for consumer testing.

Self-reported scrambled egg consumers (*n* = 109) were recruited from the NC State University staff and student population. Consumer panelists confirmed no egg food allergies and/or sensitivities prior to participation. Consumer panelists were disqualified if they were younger than 17 years of age, older than 65 years of age, or if they only consumed scrambled eggs once a month. Upon completion of the test, consumer panelists were compensated with a \$5 gift card to a local store. Compusense20 Cloud (Guelph, ON, Canada) was used for data collection and analysis. Samples were presented monadically with a 2-min enforced rest period between egg samples. Consumers evaluated various aroma, flavor, and texture liking attributes using a 9-point hedonic scale, where 1 = dislike extremely and 9 = like extremely. Consumers used a 5-point anchored Just About Right (JAR) scale to evaluate flavor and color attributes. Consumers were provided with spring water and unsalted crackers for palate cleansing.

#### *2.5. Statistical Analysis-Laying Hen Performance and Egg Lipid and Fatty Acid Content*

Each hen served as the experimental unit for all performance data. All performance data was evaluated for significance by one-way analysis of variance (ANOVA) at a significance level of *p* < 0.05 using SAS statistical software (version 9.4). If ANOVA results were significant (*p* < 0.05), Tukey's multiple comparisons t-test was conducted to compare the mean of each treatment group with the mean of every other treatment at *p* < 0.05 significance level. Comparisons were made between body weights (33 birds/treatment), feed intake (33 birds/treatment), feed conversion ratio (33 birds/treatment), and egg weights (total # of eggs collected over the 8-week feeding trial).

In total, 12 eggs per treatment (4 eggs per replicate randomly selected) were statistically analyzed by one-way ANOVA (*p* < 0.05) using SAS. Means were separated by least squares means, with Tukey-Kramer adjustment for multiple comparisons (*p* < 0.05) for treatment differences in egg quality parameters (Haugh unit, vitelline membrane strength, shell strength, yolk color score) at each bi-weekly experimental time-point (0-week, 2-week, 4-week, 6-week, 8-week). Additionally, eggs were statistically analyzed for treatment differences in egg quality parameters in all eggs collected over the 8-week feeding trial (180 total, 60 eggs per treatment). In total, 15 homogenous egg samples (5 per replicate) were statistically analyzed by one-way ANOVA (*p* < 0.05) using SAS. Means were separated by least squares means with the Tukey-Kramer adjustment for multiple comparisons (*p* < 0.05) for treatment differences in egg β-carotene content and egg lipid and fatty acid content (45 total egg samples at each time-point) weekly (2-, 3-, 4-, 5-, 6-, 7-, and 8-week).


**4.2 Social skills**

Research identifies a negative influence of parental schizophrenia on HR offspring, arising deficits in social skills in the domains of social competence, reduced smiling, affective flattening, assertion, empathy, cooperation, positive involvement, physical anhedonia, self-esteem, self-confidence, social adjustment and employment situation. This emphasises upon the debilitating negative impacts of parental schizophrenia on the psychosocial well-being of offspring.

## **4.3 Internalising and externalising behavioural problems**

Furthermore, it was found there to be a greater prevalence of internalising and externalising behavioural problems amongst HR offspring in which the incidence was greater for the HR group than the HC group [60]. HR offspring were also found to have *fewer positive emotions*, *greater negative emotions and reduced quality of life*, all of which differed from the HC group in mean scores [7]. This highlights upon the behavioural issues experienced for HR offspring further indicating a negative impact on the psychosocial well-being of HR offspring.

The findings of this review build upon the idea that parental schizophrenia influences the development of psychiatric disorders amongst HR offspring [7, 8, 10–15, 17–19, 35, 43–45, 62] which have also identified the development of psychiatric conditions amongst HR offspring. Moreover, the stated findings also build upon the idea that parental schizophrenia arises social deficits amongst HR offspring as also detailed in previous literature [7, 12, 23–28]. Overall there is a negative impact of parental schizophrenia on the psychosocial well-being of offspring as displayed across all studies. This signifies a genetic propensity being passed from the parent with schizophrenia to the offspring.

There were several interesting findings unrelated to the psychosocial well-being of HR offspring. Firstly, it was found that the prevalence for psychiatric disorders in HR offspring was higher for males than females, arising questions into potential gender characteristics, such as different coping mechanisms resulting in differed psychosocial distresses [58, 65]. The predominance of males in some studies and females in other studies may aid in differentiating the impacts of gender on the psychosocial well-being of HR offspring. Moreover, social functioning amongst HR Japanese offspring was not significantly impacted, which suggest cultural differences amongst HR offspring regarding psychosocial well-being [55]. Overall, studies included in this review cover a broad range of cultures, allowing the conclusions of this review to be generalised to many cultures, whilst building upon previous literature and highlighting upon the cumulative incidences across cultures [13–15]. Future research may look into examining western and non-western cultures to make comparisons along with the investigation of gender differences.

In addition, an anomalous finding arose, in which eight HR offspring had excellent social skills and nine HR offspring displayed below average scores, none of which were within the average on measures of social skills [61]. This suggests that offspring within this study had extreme scores on each end but may have been due to the relatively small sample size. Interestingly, it was also found that poor physical anhedonia and positive involvement were present in those aged 17 years and below, whereas those aged above 17 years displayed lower cooperation levels [63]. This highlights differences in age, in which future studies could address by comparing different age groups. Findings presented strengthen the diathesis-stress explanation. In addition, this may indicate possible ideas for intervention strategies targeted for different age groups. Therefore, these particular results can inform clinicians and therapists of the psychosocial impacts parental schizophrenia has on different age groups.

Moreover, another study found [7] lower levels of family functioning and a greater perception of losses in resources and lowered resource gains. This suggests that there are negative impacts of parental schizophrenia unrelated to the psychosocial outcomes as the environmental stressors involved in this study were lowered levels of family functioning. These environmental stressors regarding family functioning could have been maltreatment, neglect or an unidentified stressor. This finding could identify as a confounding factor, as these outcomes may have resulted in the lowered quality of life and greater negative emotions. Therefore, it may be difficult to examine a causal relation between parental schizophrenia and the impact this has on the psychosocial well-being of HR offspring. Future research may consider investigating these aspects to distinguish the extent to which this impacts HR offspring. It was also found that those within the HR group had higher scores on measures of quality of life than the ME and FE group [56]. This suggests that there are confounding factors influencing the findings in an opposite direction. Highlighting upon the existence of possible confounding factors, there was a greater prevalence of psychiatric disorders amongst HR offspring in disturbed familial environments [66]. This implies that the environmental factors surrounding HR offspring may play an important role in the development of negative impacts on the psychosocial well-being of HR offspring.

**175**

*The Impacts of Parental Schizophrenia on the Psychosocial Well-Being of Offspring:…*

Environmental factors are important to identify, as these alone can result in negative outcomes such as the incidence of psychiatric conditions amongst HR offspring. This is because the offspring may experience an upbringing whereby the environment is distorted, thus elevating the risk for negative impacts on the psychosocial well-being of offspring. Across all findings, the effect sizes and mean differences between the HR groups and control groups were not 100%, suggesting other factors influencing the onset of negative psychosocial distress and difficulties. Also, there were not vast differences between the HR group and control groups regarding the effect sizes and mean differences, for example, it was found there to be mean incidence of 39.15 for externalising/internalising behavioural issues amongst HR offspring, in comparison to a 31.53 mean incidence for the HC group [60]. This supports the idea that the environmental factors play a role in the negative impacts, further strengthening the idea of a diathesis-stress explanation. The direction of the findings may have been due to the participant size across studies. Most studies included had a relatively large sample size [56–59, 62, 63, 65, 66]. However, some studies had a relatively small sample size. The small sample size amongst these particular studies may have resulted in the effect size and mean incidence scores/ratings produced and thus reducing the statistical power and generalisability of these findings. Therefore, future reviews may aim to include studies consisting of a relatively larger sample, similar to each other and the control groups,

There were many confounding factors which were not mentioned or described such as environmental factors, including maltreatment, child abuse, marital conflict and other stressful life events as detailed in previous literature [12, 34, 44–49, 51]. This may imply that poor psychosocial functioning may not be due to having a parent with schizophrenia but rather the surrounding environment of the offspring. Previous literature identifies a link to the negative impacts on the psychosocial wellbeing of HR offspring which may suggest and support the diathesis-stress model of the development of these outcomes. This model suggests that environmental factors increase the risk of developing psychological difficulties, particularly amongst HR offspring as they have a genetic vulnerability as mentioned in previous literature [36–42] in which an environmental stressor occurs which triggers a negative outcome (i.e. the onset of a psychiatric condition or symptom), also stated in previous literature [43, 44]. To summarise, findings cannot establish which factor causes the outcome; therefore, future research must aim to address this by incorporating the

Moreover, findings revealed there to be a positive correlation between the duration of parental schizophrenia on the self-esteem of HR offspring [64] which suggests that the longer the parent was diagnosed with schizophrenia, the greater the self-esteem scores within the HR group (mean duration of parental illness = 11.67 years). This is a confounding result, as it implies that parental schizophrenia in the long-term had an almost reversal effect on scores of levels of self-esteem. This may have been due to the HR offspring being able to adjust and adapt to their parent's mental illness over time, which also further suggests confounding factors involved in this outcome. All other studies included did not sufficiently state the duration of parental illness or age of offspring at which parent diagnosis occurred. This is an important aspect for future studies to address.

Furthermore, it was also found that HR offspring had lower self-esteem in earlyonset offspring, in comparison to later onset offspring in which self-esteem scores were higher (mean age of onset = 10.6 years) [64]. This indicates that the younger the offspring is when their parent is diagnosed, the worse the impact this has on

*DOI: http://dx.doi.org/10.5772/intechopen.91658*

**4.4 Confounding factors**

to prevent biased findings.

measures for environmental factors.

*The Impacts of Parental Schizophrenia on the Psychosocial Well-Being of Offspring:… DOI: http://dx.doi.org/10.5772/intechopen.91658*

#### **4.4 Confounding factors**

*Quality of Life - Biopsychosocial Perspectives*

The findings of this review build upon the idea that parental schizophrenia influences the development of psychiatric disorders amongst HR offspring [7, 8, 10–15, 17–19, 35, 43–45, 62] which have also identified the development of psychiatric conditions amongst HR offspring. Moreover, the stated findings also build upon the idea that parental schizophrenia arises social deficits amongst HR offspring as also detailed in previous literature [7, 12, 23–28]. Overall there is a negative impact of parental schizophrenia on the psychosocial well-being of offspring as displayed across all studies. This signifies a genetic propensity being

There were several interesting findings unrelated to the psychosocial well-being of HR offspring. Firstly, it was found that the prevalence for psychiatric disorders in HR offspring was higher for males than females, arising questions into potential gender characteristics, such as different coping mechanisms resulting in differed psychosocial distresses [58, 65]. The predominance of males in some studies and females in other studies may aid in differentiating the impacts of gender on the psychosocial well-being of HR offspring. Moreover, social functioning amongst HR Japanese offspring was not significantly impacted, which suggest cultural differences amongst HR offspring regarding psychosocial well-being [55]. Overall, studies included in this review cover a broad range of cultures, allowing the conclusions of this review to be generalised to many cultures, whilst building upon previous literature and highlighting upon the cumulative incidences across cultures [13–15]. Future research may look into examining western and non-western cultures to

passed from the parent with schizophrenia to the offspring.

make comparisons along with the investigation of gender differences.

psychosocial impacts parental schizophrenia has on different age groups.

perception of losses in resources and lowered resource gains. This suggests that there are negative impacts of parental schizophrenia unrelated to the psychosocial outcomes as the environmental stressors involved in this study were lowered levels of family functioning. These environmental stressors regarding family functioning could have been maltreatment, neglect or an unidentified stressor. This finding could identify as a confounding factor, as these outcomes may have resulted in the lowered quality of life and greater negative emotions. Therefore, it may be difficult to examine a causal relation between parental schizophrenia and the impact this has on the psychosocial well-being of HR offspring. Future research may consider investigating these aspects to distinguish the extent to which this impacts HR offspring. It was also found that those within the HR group had higher scores on measures of quality of life than the ME and FE group [56]. This suggests that there are confounding factors influencing the findings in an opposite direction. Highlighting upon the existence of possible confounding factors, there was a greater prevalence of psychiatric disorders amongst HR offspring in disturbed familial environments [66]. This implies that the environmental factors surrounding HR offspring may play an important role in the development of negative impacts on the psychosocial well-being of HR offspring.

In addition, an anomalous finding arose, in which eight HR offspring had excellent social skills and nine HR offspring displayed below average scores, none of which were within the average on measures of social skills [61]. This suggests that offspring within this study had extreme scores on each end but may have been due to the relatively small sample size. Interestingly, it was also found that poor physical anhedonia and positive involvement were present in those aged 17 years and below, whereas those aged above 17 years displayed lower cooperation levels [63]. This highlights differences in age, in which future studies could address by comparing different age groups. Findings presented strengthen the diathesis-stress explanation. In addition, this may indicate possible ideas for intervention strategies targeted for different age groups. Therefore, these particular results can inform clinicians and therapists of the

Moreover, another study found [7] lower levels of family functioning and a greater

**174**

Environmental factors are important to identify, as these alone can result in negative outcomes such as the incidence of psychiatric conditions amongst HR offspring. This is because the offspring may experience an upbringing whereby the environment is distorted, thus elevating the risk for negative impacts on the psychosocial well-being of offspring. Across all findings, the effect sizes and mean differences between the HR groups and control groups were not 100%, suggesting other factors influencing the onset of negative psychosocial distress and difficulties. Also, there were not vast differences between the HR group and control groups regarding the effect sizes and mean differences, for example, it was found there to be mean incidence of 39.15 for externalising/internalising behavioural issues amongst HR offspring, in comparison to a 31.53 mean incidence for the HC group [60]. This supports the idea that the environmental factors play a role in the negative impacts, further strengthening the idea of a diathesis-stress explanation.

The direction of the findings may have been due to the participant size across studies. Most studies included had a relatively large sample size [56–59, 62, 63, 65, 66]. However, some studies had a relatively small sample size. The small sample size amongst these particular studies may have resulted in the effect size and mean incidence scores/ratings produced and thus reducing the statistical power and generalisability of these findings. Therefore, future reviews may aim to include studies consisting of a relatively larger sample, similar to each other and the control groups, to prevent biased findings.

There were many confounding factors which were not mentioned or described such as environmental factors, including maltreatment, child abuse, marital conflict and other stressful life events as detailed in previous literature [12, 34, 44–49, 51]. This may imply that poor psychosocial functioning may not be due to having a parent with schizophrenia but rather the surrounding environment of the offspring. Previous literature identifies a link to the negative impacts on the psychosocial wellbeing of HR offspring which may suggest and support the diathesis-stress model of the development of these outcomes. This model suggests that environmental factors increase the risk of developing psychological difficulties, particularly amongst HR offspring as they have a genetic vulnerability as mentioned in previous literature [36–42] in which an environmental stressor occurs which triggers a negative outcome (i.e. the onset of a psychiatric condition or symptom), also stated in previous literature [43, 44]. To summarise, findings cannot establish which factor causes the outcome; therefore, future research must aim to address this by incorporating the measures for environmental factors.

Moreover, findings revealed there to be a positive correlation between the duration of parental schizophrenia on the self-esteem of HR offspring [64] which suggests that the longer the parent was diagnosed with schizophrenia, the greater the self-esteem scores within the HR group (mean duration of parental illness = 11.67 years). This is a confounding result, as it implies that parental schizophrenia in the long-term had an almost reversal effect on scores of levels of self-esteem. This may have been due to the HR offspring being able to adjust and adapt to their parent's mental illness over time, which also further suggests confounding factors involved in this outcome. All other studies included did not sufficiently state the duration of parental illness or age of offspring at which parent diagnosis occurred. This is an important aspect for future studies to address.

Furthermore, it was also found that HR offspring had lower self-esteem in earlyonset offspring, in comparison to later onset offspring in which self-esteem scores were higher (mean age of onset = 10.6 years) [64]. This indicates that the younger the offspring is when their parent is diagnosed, the worse the impact this has on

HR offspring's self-esteem levels. This further indicates that age has an impact on the vulnerability of HR offspring, in which developmental literature can address in future research, by conducting more longitudinal studies to map the trajectory of this vulnerability and make comparisons between different age groups (i.e. less than 10 years old and greater than 10 years old). Moreover, parental physical illness was incorporated by eliminating HR offspring of those that were suffering from chronic physical illnesses [64], to minimise the effect of this stressor. This is because this may cause greater significant stress on the HR offspring, which may impact the psychological difficulties the HR offspring may encounter. Therefore, to minimise bias amongst the findings, these particular HR offspring were excluded from the study.

Considering living arrangements amongst HR offspring, the HR perspective was explored amongst those adopted offspring living away from the biological parent suffering from schizophrenia [66]. Findings revealed that HR offspring still displayed a greater prevalence of psychopathology in comparison to the HC group. This finding highlights upon a predominant genetic vulnerability amongst HR offspring, despite being reared away from their biological parent with schizophrenia. This builds upon previous literature [36–42] highlighting upon the idea that brain abnormalities are genetically transmitted to HR offspring, thus supporting a diathesis-stress explanation.

#### **4.5 Clinical implications**

The findings build upon the idea that parental schizophrenia negatively impacts the psychosocial well-being of HR offspring regarding the following aspects: the prevalence of emotional and behavioural symptoms, internalising and externalising behaviours, psychiatric conditions, lowered quality of life, poorer self-concept, social adjustment, social functioning and employment situation. The greatest impact across studies in this review was the impairments in social functioning, which can be interpreted using the diathesis-stress explanation. The genetic vulnerabilities appeared to have been transmitted from parent to offspring, whereby environmental factors and stressors surrounding these individuals triggered an onset of these impairments. This review provides a more current set of findings with the inclusion of social impacts as well as psychological impacts, in comparison to the previous systematic review based solely upon the development of psychiatric conditions of HR offspring [11].

The current findings can aid public health authorities in raising awareness for this HR population by means of prioritising education in mental health, mass media and by increasing public funding. Revelations can also encourage early visits to mental health centres for screenings and evaluations to prevent and provide support [12]. It can also inform clinicians about targeted intervention strategies for HR offspring, such as taking into consideration the developmental differences in the onset of psychosocial distress. All these aspects can either prevent the later emergence of psychosocial difficulties or reduce experienced.


**1. Introduction**

294 Chromatography – The Most Versatile Method of Chemical Analysis

*Crystallogr D*. 2010; 66:12-21.

*Crystallogr D*. 2010; 66:486-501.

*Nuc Acids Res*. 2004; 32:W665-W667.

sequencing. *Biochem J*. 1996; 318:989-996.

*USA*. 2008.

10041.

125.

(5):355-364.

[55] Gilkes NR, Kilburn DG, Miller RC, Warren RAJ. Structural and functional-analysis of a

[56] Goujon M, McWilliam H, Li W, Valentin F, Squizzato S, Paern J, et al. A new bioinformatics analysis tools framework at EMBL-EBI. *Nuc Acids Res*. 2010; 38:W695-9. [57] Arnold K, Bordoli L, Kopp J, Schwede T. The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. *Bioinformatics*. 2006; 22:195-201. [58] Chen VB, Arendall III WB, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, et al. MolProbity: all-atom structure validation for macromolecular crystallography. *Acta*

[59] Emsley P, Lohkamp B, Scott W, Cowtan K. Features and development of Coot. *Acta*

[60] DeLano WL. *The PyMOL molecular graphics system, Delano Scientific LLC, Palo Alto, CA -*

[61] Dolinsky TJ, Nielsen JE, McCammon JA, Baker NA. PDB2PQR: an automated pipeline for the setup, execution, and analysis of Poisson-Boltzmann electrostatics calculations.

[62] Baker NA, Sept D, Joseph S, Holst MJ, McCammon JA. Electrostatics of nanosystems: application to microtubules and the ribosome. *Proc Natl Acad Sci USA*. 2001; 98:10037-

[63] Janecek S, Sevcik J. The evolution of starch binding domain. *FEBS Lett*. 1999; 456:119-

[64] Iefuji H, Chino M, Kato M, Limura Y. Raw-starch digesting and thermostable alphaamylase from yeast *Cryptococcus* sp. S2: purification, characterization, cloning, and

[65] Hostinová E, Janeček Š, Gašperík J. Gene sequence, bioinformatics and enzymatic characterization of α-amylase from *Saccharomycopsis fibuligera* KZ. *Prot J*. 2010; 29

bacterial cellulase by proteolysis. *J Biol Chem*. 1989; 264 (30):17802-17808.

For successful fertilization, motility is the most obvious and most essential sperm function and has been repeatedly shown to be predictive of fertilization in vitro [1]. Several studies have shown that the motility characteristics of spermatozoa are of the utmost importance for the men's fertility [2]. Spermatozoa dysfunction is the single most important cause of infertility. A decrease in spermatozoa motility with time is universal phenomenon. This reduction differs from species to species and also among individuals of the same species, as in the human male. Most investigators agree that the majority of spermatozoa cease to move within the first 24 hours. The survival of spermatozoa after ejaculation is dependent on the environmental conditions under which they are kept. In the female genital tract they may remain active for several days [3], but their activity is of much shorter duration if they remain in the seminal fluid outside the body.

Male genital infections are relevant cause in the etiology of infertility due to abnormalities in sperm quality [4,5], affecting spermatozoal motility. The comparison of semen characteristics between infected and non-infected men show that motile spermatozoa are lower when the microorganisms are present in the semen [6]. It appears that bacteria have a direct effect on sperm motility with negative consequences in fertility. Among bacterial species that interact with spermatozoa are well-known causative pathogens of genitourinary infections such as *Escherichia coli, Ureaplasma urealyticum, Mycoplasma hominis, Chlamydia trachomatis* [7]. Of the various bacteria, *E. coli* is the most extensively studied microorganism in relation to infertility as a result of interaction with spermatozoa [8]. It is also the primary bacteria associated with prostatitis and epididymitis [9]. Several authors describe sperm

© 2012 Prabha and Kaur, licensee InTech. This is an open access chapter 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. © 2012 Prabha and Kaur, licensee InTech. This is a paper 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.

agglutination and immobilization by *E. coli* [10,11]. Paulson & Polakoski [12] investigated the mechanism of how *E. coli* immobilizes spermatozoa and they reported a factor, apparently excreted by the bacteria which immobilizes spermatozoa without agglutinating it. However, Diemer et al. [13] reported that *E. coli* inhibits sperm motility by directly adhering to and agglutinating spermatozoa. Rapidity and extent of sperm–*E. coli* agglutination indicated strong adhesive forces. Bartoov et al*.* [8] proposed that mannose plays a critical role in adherence of *E. coli* to sperm. Although, a number of studies have evaluated the ability of *E. coli* to affect sperm motility by adherence, agglutination and dialyzable factors, however, none have identified the exact mechanism of interaction between spermatozoa and bacteria.

Isolation and Purification of Sperm Immobilizing/Agglutinating Factors from Bacteria and Their Corresponding Receptors from Human Spermatozoa 297

supernatant failed to do so indicating that the agglutination of sperm might be associated with bacterial cells and not their metabolites. Pretreatment of *S. aureus* by sonication produced bacterial fragments that were unable to agglutinate sperm. Centrifugation of *S. aureus* fragments at 10,000 g for 5 min did not eliminate sperm agglutinating elements from the solution, indicating the sperm agglutinating factor to be present in sonicated supernatant. Based on its sperm agglutinating activity, the bioactive molecule from the sonicated supernatant was purified by ammonium sulphate precipitation, gel permeation chromatography and ion exchange chromatography. The sonicated supernatant was fractionated with ammonium sulphate so as to get 20, 40, 60 80 and 100% saturation. The flasks were kept at 4°C overnight and next day the precipitates were collected by centrifugation at 10,000 g for 15 min at 4°C and were redissolved in minimum amount of PBS. SAF was precipitated by ammonium sulphate at 40% saturation. The protein was dialyzed in preactivated dialysis bags against PBS under cold conditions and concentrated against polyethylene glycol (PEG) 6000 at 4°C. Further purification was done using Sephadex G-200 and DEAE cellulose column chromatography. The fractions containing approximately 1mg protein were applied on Sephadex G-200 (Pharmacia Fine Chemicals, Uppsala), column (2 cm X 31 cm) equilibrated and eluted with PBS. The aspirator bottle with PBS was joined with a fine capillary tube to maintain constant head pressure and allowed to run for 24 h. Fractions of 3 ml each were collected and each fraction was read at 280 nm on U.V. spectrophotometer. The fractions showing spermagglutinating activity i.e. fraction (4-7) with a peak value in fraction 5 were pooled and concentrated against PEG 6000 (Figure 1a) and applied to DEAE cellulose (Hi Media Laboratories Ltd., Mumbai, India) column. A column of 2 cm x 15 cm was made with activated slurry of DEAE cellulose, an anion exchanger with a pressure of about 1 cm water/cm of height of gel bed. The column was washed with PBS until the ion exchanger reached ionic equilibrium with the starting buffer. The aspirator bottle with PBS was joined with a fine capillary tube to maintain a constant head pressure and allowed to run. Final elution was done with PBS containing 0.05, 0.1, 0.2, 0.4 and 0.6 M NaCl. Fractions of 4 ml each were collected and read at 280 nm on U.V. spectrophotometer (Hitachi U-2900). All fractions were again checked for agglutination of spermatozoa. The fractions showing agglutination of sperm (35-39) were pooled and concentrated (Figure 1b). The purified pooled fractions were dialyzed, lyophilized and then subjected to protein estimation. The purification status of SAF was checked by gel electrophoresis (PAGE). Molecular weight estimation was done by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) using a 12% resolving gel containing SDS [17]. 10µl of the pooled and concentrated sample obtained from DEAE cellulose column was mixed with loading dye (Bromophenol blue: glycerol) in the ratio of 1:50. The sample was applied to the gel and subjected to a current of 15 mA and run initially at 50V and thereafter at 100V. The gel was visualized by staining with Coomassie Brilliant Blue R-250. The

molecular weight of SAF was estimated to be approximately, 65 kDa (Figure 1c).

*S. aureus*

**4. Extraction and purification of sperm immobilization factor (SIF) from**

For extraction of SIF, *S. aureus* was grown in Brain Heart Infusion (BHI) broth for 72 h at 37°C under shaking conditions (150 rpm). The cell culture was subjected to centrifugation at

In addition to *E. coli*, *Staphylococcus aureus,* the predominant flora in infertile men, has also been reported to cause a significant decrease in sperm motility [14]. Emokpae et al. [15] while studying the contribution of seminal tract infection to sperm density, asthenozoospermia and teratozoospermia, observed *S. aureus* as the causative organism accounting for 68.2% of seminal infections. Most practitioners dismiss this infection as mere contamination, which is assumed to have no significance. Semen that passes through the genital tract is routinely contaminated with staphylococci. However, since the prevalence of abnormal sperm indexes is high, it was suggested that *S. aureus* infection should be treated and no longer ignored when managing male factor infertility [15]. *S. aureus* is known to produce various toxins and enzymes that may be exerting damaging effect on human sperm, but its mechanism of action also needs further investigation. Therefore, the present work was undertaken to study the mechanism of immobilization/agglutination of spermatozoa by *S. aureus* and *E. coli*.


**3.3. Providing clinical information for the therapist**

The therapist needs information about the patient to plan the therapeutic process and interventions adequately. The first phase of CBT includes individual case formulation and clinical diagnosis. In CBT, several ways of obtaining information about the patient are possible, for instance, clinical interview, and self-observational methods [28]. An additional way of gaining information about the patient is to ask him/her about his/her dream content [26].

The approach of finding relationships between dreaming and waking experiences, which is typical of the therapeutic setting, starts with a dream and goes into its connections with the waking-life issues of the patient. Studies on dreams in psychodynamic therapy have shown that they provide information about, among others, patient's patterns of interpersonal functioning, his/her personality structure, and images of self and others. Dreams can also reveal information the patient is not aware of, for instance, his/her attitude toward the therapist [26, 33]. Important information for the therapist in CBT that may be provided by dreams concerns the patient's cognitive patterns [9], core beliefs [2], schemas [24], cognitive distortions [2], patterns of behaviors [15], patterns of affective responses [23], and resources [2].

Often dreams quickly and efficiently reveal information that is unique to the patient, especially information that the patient is not aware of or just does not want to share with the therapist. When the specialist recognizes this information, he/she can help the patient perceive it and subsequently change maladaptive cognitive and behavioral patterns. Dreams can also contain crucial information about a patient's emotions [26].

Dreams can reveal much important information about the patient to the therapist. Working with dreams in therapy may facilitate the speed at which some personal material is introduced to collaborative therapeutic discussion, helps the patient recognize and express emotions and thoughts, and may even shorten therapy [26]. However, there is no research on this use of dreams related to providing clinical information to the therapist in CBT.


**2.3 Case 3**

A 79-year-old man present with bullous keratopathy in his right eye. He underwent traumatic cataract extraction and BDI lens implantation in 2003. On examination, his bestcorrected visual acuity was hand motion in the right, and the IOP was within the normal limits without medication. DSAEK was performed using a neonate donor. The graft markedly contracted during the first postoperative week. Two months later, the patient received a second surgery to exchange the graft. No postoperative complications occurred this time. The last follow-up visit was 3 month after the second DSAEK, the visual acuity improved to 0.1. IOP was good.

The DSAEK basic procedures were same with the reported by Terry. The implants was prepared by using a Moria (Antony, France) automated microkeratome and a Moria artificial chamber.

Fig. 3. (case 3) Slit lamp photograph (A) and AS-OCT (B) shows the donor tissue shrink during the first postoperative week. (C) and (D) shows the newly exchanged donor disc is well attached.


**9. Conclusion**

Since its first description in 1992, TPVL has undergone an important evolution and become a confirmed technique. It is currently considered as a first-line and vital solution for PVLs reduction by many teams, even if surgery remains the reference technique in guidelines. It is important to note that it demands high expertise and is feasible only in Ref. centers with a multidisciplinary team contribution. It remains limited by dedicated devices availability and lack of financial support.