Characteristics of Animals under Specific Environmental Conditions

### **Chapter 4**

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

*Gbolabo Olaitan Onasanya,*

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

#### **Abstract**

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

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

#### **1. Introduction**

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

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

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

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

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

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

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

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

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

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

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

#### **Figure 2.**

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

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

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

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

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

From the previously published study, 70 animals were placed into seven groups with 10 individuals each. In total, 70 Indian Zebu-Jersey crossbred cattle breed's blood samples yielded 70 aliquots of PBMCs, both stressed and unstressed cells. With the exception of the 0°C TAC, the PBMCs were exposed to each of the four TACs for 3 h and 6 h (0, 37, 40, and 45°C) (**Figure 2**). Before the TSS procedure, the number of viable cells were estimated to be between 7.04 <sup>10</sup><sup>6</sup> -2.56 <sup>10</sup><sup>7</sup> cells/mL. About 1 <sup>10</sup><sup>6</sup> PBMCs/mL were present in each aliquot of 500 <sup>μ</sup>L [1].

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

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

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

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

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

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

**Figure 3.**

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

Maryland, USA) (CELESTRON Labs CB2000C Compound Microscope, Celestron, LLC., California, USA) (**Figure 2a**). Before TSS, it was estimated that there were 7.04 � <sup>10</sup><sup>6</sup> to 2.56 � <sup>10</sup><sup>7</sup> viable cells per millilitre. Total RNA was extracted from the isolated PBMCs. Using a Thermo-Scientific-Nano Drop 2000 spectrophotometer, total RNA was quantitated (Shimadzu co-operation, Kyoto, Japan).

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

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

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

Average number of viable PBMCs per square

<sup>¼</sup> Total number of viable PBMCs in 4 Squares*=*<sup>4</sup> (2)

Dilution factor <sup>¼</sup> Total volume Volume of PBMCs <sup>þ</sup> Volume of trypan blue dye*=*Volume of PBMCs

(3)

Concentration of Viable PBMCs*=*Square <sup>¼</sup> Average number of viable PBMCs � Dilution factor � <sup>10</sup><sup>4</sup> (4)

For this study, two dilution factors were used.

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

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

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


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

#### **Table 1.**

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

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

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

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

How to guarantee that each treatment group has equal number of PBMCs is shown in **Table 2**. For instance, the maximum PBMC count in the four treatment groups is 3.08 �10<sup>7</sup> . Note that, the treatment group with the highest PBMC count will be used as the benchmark for the computation to guarantee that PBMC counts are equal across treatment groups. Eqs. (5–7) demonstrate the various equations to make the estimations.

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

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

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

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

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



**Table 2.**

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

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


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

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

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

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

#### **13. Conclusion**

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

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


#### **Table 3.**

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

### **Acknowledgements**

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

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Gbolabo Olaitan Onasanya1,2,3\*, Aranganoor Kannan Thiruvenkadan2 , Alice Adishetu Yisa4 , Krishnaswamy Gopalan Tirumurugaan<sup>3</sup> , Murali Nagarajan<sup>2</sup> , Saravanan Ramasamy<sup>2</sup> , Raja Angamuthu<sup>5</sup> , George Mutani Msalya<sup>6</sup> and Christian Obiora Ikeobi<sup>7</sup>

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

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

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

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

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

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

7 Federal University of Agriculture Abeokuta, Abeokuta, Nigeria

\*Address all correspondence to: onasanya.gbolabo@gmail.com

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

### **References**

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

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

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

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

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

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

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

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

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

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

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

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

#### **Chapter 5**

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

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

#### **Abstract**

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

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

#### **1. Introduction**

Puberty corresponds to the transition into adulthood and can be defined in rams as the time when fertile spermatozoa are present in the ejaculate [1]. For some authors it is defined as the time when rams show 'interest' in females in estrus by successive mounting with ejaculation [2]. It is also described as the stage of sexual maturation when a ram can display complete sexual behaviour, to produce and to release gametes [3]. Another definition of puberty in ram lambs is based on a sustained rise in plasma testosterone concentrations over three consecutive blood samplings performed in one week and confirmed by the presence of spermatozoa in the ejaculate with at least 30% mass motility [1, 4]. In general, sexual development of ram lambs appears to be more closely associated with body growth than with chronological age. Body weight can be a good criterion for the achievement of puberty than chronological age alone [2]. Rate of testis growth is reported to be more rapid in lambs of highly prolific breeds such as the Finnish Landrace than in less prolific breed [5]. Louda et al, after observing testis growth and small but consistent differences in the development of sexual activity and of sperm production, suggested that young rams of prolific breeds (Romanov and Finnish Landrace) may differ in their future reproductive performance. The early pubertal development associated with increased body weight is desirable in terms of improved reproductive performance [5, 6]. Broad information about the onset of puberty is of considerable importance for successful reproductive management [7]. On one hand, it is important to avoid inbreeding by separating prepubertal rams from ewes, especially in counties with extensive breeding practices and poor separation between animal groups of different ages or different reproduction status. On the other hand, insufficient sexual maturity of young rams may lead to reduced flock fertility. A few information is available in terms of testosterone levels, semen quality, libido and sexual performance at high altitude conditions in a non-seasonal country [8].

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

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

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

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

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

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

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

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

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

The study was conducted between July 2012 and April 2013 at the experimental farm of the National University of Colombia at 2650 m of altitude at 4°42<sup>0</sup> latitude north and 74°12<sup>0</sup> longitude west, near Bogotá (Colombia).

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

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

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

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

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

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

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

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

#### **2.4 Body measurements**

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

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

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

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

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

#### **4. Results**

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

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

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

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

#### **5. Discussion**

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


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

#### **Figure 1.**

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


#### **Table 2.**

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


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

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

#### **Table 3.**

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

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

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

#### **Figure 2.**

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

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

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

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

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

#### **Author details**

Harvey Lozano<sup>1</sup> \*, Jimmy Vargas<sup>2</sup> , Liliana Chacón<sup>3</sup> and Nathalie Kirschvink<sup>4</sup>

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

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

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

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

\*Address all correspondence to: hlozanoma@unal.edu.co

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

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

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

*Rita Payan Carreira, Veterinary Medicine and Science Series Editor*

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Breeding Strategies for Healthy and Sustainable Development of Animal Husbandry

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Breeding Strategies for

Healthy and Sustainable

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*Edited by Xiaojun Liu and Hong Li*