Genetic parameter estimation of semen characteristics and sire selection traits for Bonga sheep under the community-based breeding program

Abstract Pursuing the community-based breeding program (CBBP) selection of the breeding sire for Bonga sheep estimated breeding value by six months body weight, testicle size, and physical characteristics were used as selection criteria. A study was conducted to investigate the association of sire selection traits with semen characteristics and to estimate their heritability. Data were collected from a total of 101 adult breeding sires of Bonga sheep. Semen was collected using artificial vagina and processed for semen qualities. The variance components and genetic parameters were estimated using multivariate analysis of animal model using restricted maximum likelihood method of WOMBAT software. Six different animal models were compared for the dataset and best-fit model was selected using Akaike information Criteria (AIC). Direct additive effect (model 1) was the best-fit model for the current dataset. The direct heritability for studied traits were 0.23 ± 0.122, 0.13 ± 0.129, 0.13 ± 0.110, 0.09 ± 0.036, 0.37 ± 0.155, and 0.11 ± 0.162 for semen volume, mass motility, concentration, total spermatozoa, six months body weight, and scrotum circumference, respectively. Moderate heritability of six months body weight and semen volume indicated that further breed improvement through selection is possible. Low heritability of semen characteristics traits indicated that good semen quality could be obtained by improving environmental management of the sire. Both six months body weight and scrotum circumference have high and positive correlation with sperm mass motility, and concentration but negatively correlated with semen volume and total spermatozoa production. Given the strong correlation between semen characteristic qualities, the associated response was anticipated.

circumference have high and positive correlation with sperm mass motility, and concentration but negatively correlated with semen volume and total spermatozoa production.Given the strong correlation between semen characteristic qualities, the associated response was anticipated.

Introduction
Small ruminants are extremely important in the livelihoods of smallholder farmers in Ethiopia.This sector could easily be improved with targeted interventions on the most limiting factors within the value chain (Haile, 2017).The success of a small ruminant operation depends on the number of lambs and kids raised, weaned, and marketed each year.
The reproductive capacities of sires are directly or indirectly involved in the reproductive process, either during natural reproduction or by the production of semen used for artificial insemination (Allaoui et al., 2014).Sire selection that can settle ewes and genetic potential for rapid growth enhance farm productivity (Pezzanite et al., 2019).Sires contribute up to 75% of the genetic change in a flock; therefore, it is important to not shortcut the selections or care of them.
To identify sires that are capable or not for servicing of females, producers should perform breeding sound examinations (BSE).The BSE consists of a physical examination, inspection of the reproductive organs, and semen collection and evaluation (Pezzanite et al., 2019).The BSE of breeding sires should incorporate origin and breeding value, screening for sexually transmitted diseases, physical and reproductive organs examination, and sperm production and viability serving capacity (Rekik, 2016).
Measurement of scrotum circumference (SC) reflects the weight of the gonad and the ability of sperm production (Salhab et al., 2003).It has great value as indicators of the onset of puberty, total semen production, semen quality, pathological conditions of testes, and potential infertility.It has an ability for genetic improvement of fertility in ram lambs than any other semen characteristics (Rege et al., 2000).It is a highly heritable trait that is considered an excellent indicator of sperm production in the sire (Moghaddam et al., 2012).The males with larger testes have higher daily sperm output and tend to sire daughters that reach puberty at an earlier age and can ovulate more ova during each estrus period (Söderquist & Hultén, 2006).Positive associations between sires with high scores for sexual performance and ewe fertility have been reported (Perkins & Fitzgerald, 1992).
The components of semen quality evaluation include semen volume, sperm concentration, sperm motility, and the percent morphologically normal sperm.Among the male factors affecting artificial insemination for sheep were age of the ram and its sperm mass motility which is predictive of the fertility (David et al., 2015).
Bonga sheep is the largest indigenous sheep breed in Ethiopia and is subjected to genetic improvement through a community-based breeding program (CBBP) (Areb et al., 2021).The selection and dissemination of sires in CBBP were based on physical evaluation, estimated breeding value by their six months' body weight, and pedigree information.These methods do not guarantee the productivity and fertility of sires for use and dissemination purposes.Many males that do not meet the requirements set for the BSE may not be sterile (Van Metre et al., 2012).During selection, candidates that have small testicle sizes were culled even they full filed requirement of selection criteria.
Currently, an artificial breeding program for sheep is an emerging technology in Ethiopia.Understanding ram-side factors, such as the fertilizing potential of an ejaculate, is crucial which may contribute to variability in conception outcomes.There is no information on the association of selected body weight with testicle size, semen volume, sperm mass motility, and concentration.Heritability and genetic correlation estimation for semen quality and quantity could be important to determine future breed improvement efforts for Ethiopia sheep industry.
Therefore, this study aimed to quantify the genetic association and heritability of semen characteristics namely semen volume, mass motility, concentration, and sire selection traits, namely, six-month body weight, and scrotum circumference.

Description of study area
The study was conducted in two districts, namely Adiyo kaka, and Tello, in the Kaffa zone of the southwest regional state of Ethiopia.This zone is situated in the 7º 34´N latitude and 37º 6´E longitude, and is 467 km away from the capital city; Addis Ababa.The area is characterized by a mixed crop-livestock production system.It has one major rainy season that extends from May to October, and the dry season lasts from October to April (Mirkena et al., 2012).The altitude range of the study area is 1600 to 3348 meters above sea level and the minimum and maximum temperature were 14 and 32°C, respectively, with an average of 24°C.Such updated climatic condition from each district is not available, thus such climatic information (mean maximum and minimum temperature http://climexp.knmi.nl/select.cgi?field=cru4_tmp) was obtained from online source, namely, climatic explorer information of KNMI (Royal Netherland Meteorological Institute national research and information center for meteorology) but the altitude was simple GPS data.

Experimental animals
A total of 101 experimental adult breeding sires were selected from the CBBP of Bonga sheep.In this breeding program, the pedigree, growth, and reproductive performance data were recorded after identification by ear tag of the animal.Selection of breeding sire was done at six months of age.Estimated breeding value at six months of age, physical characteristics, free from any defect and testicular sizes are the criteria for selection of breeding sires.During the selection of sires, the physical BSE was undertaken, namely, availability of cryptorchidism, defect of leg, feet, teeth, eye, movability of testicle within the scrotum, availability of firmness, abscess and swelling of testicle and epididymis.The objective of the BSE is to evaluate a male based on his ability to produce and deliver semen in sufficient quantity and quality and achieve a high conception rate.

Data collection and analysis
Sires' pedigree information and six months' body weight data were taken from the CBBP dataset of Bonga sheep.The data structure and the number of records for the studied traits were presented in Table 1.The number of sire and dams for the selected breeding sires were 30 and 39, respectively.Scrotum circumference was measured using measuring tape on the widest part of the scrotum in centimeters.Semen was collected from each selected sire using an artificial vagina (AV) with a temperature of 42-43ºC.Prior to collection, the prepuce was cleaned to prevent contamination of the semen.Semen volume was recorded using a graduated collecting glass with 0.1 ml accuracy.
Measurement of the sperm concentration was done using a portable spectrophotometer pre-calibrated for ram semen (ovine-caprine accuread photometer; IMV®, France).Sperm cell concentration was estimated after taken of 10 µl of fresh semen using a micropipette and putting on the 4 ml of normal saline (0.9%) on the UV Macrocell (UV Macro Cell 2.5 to 4.5 ml, Great Britain) and mixing gently and measure the concentration using AccuRead IMV Technologies SA, 232 Spectrophotometer.Sperm mass motility was estimated subjectively using a light microscope.The mass motility was graded according to (Macfarlane, 1991).
Genetic parameters for semen characteristics were estimated using multivariate analysis of the restricted maximum likelihood method with an animal model using WOMBAT software (Meyer, 2007).Six different types of models were used for genetic parameter estimation that addresses additive effect of animal (Z 1a ), maternal effect (Z 2 m ), and maternal permanent environmental effect (Z 3c ).The fixed effects included in the model were sire birth years, birth type, sire location, sire body condition score, birth season, and dam parity of birth.
The models are as follows: where Y = semen characteristics traits, b = a vector contained fixed effects (sire birth year, birth type, birth season, location, body condition score, and dam parity), a = direct additive genetic effects, m = maternal genetic effects for lamb traits, c = maternal permanent effect, e = residual effect, X= incidence matrices observations to b, Z 1 = incidence matrices to a, Z 2 = incidence matrices to m, Z 3 = incidence matrices to c, A = numerator relationship matrix between animals, and σ am = covariance between direct and maternal genetic effects.
Estimates of additive direct (h 2 a ) and additive maternal (h 2 m ) heritability and the ratio of maternal permanent environmental variance with phenotypic variance (c 2 ) were calculated as ratios of estimates of additive direct (σ a  variances to the phenotypic variance (σ p 2 ), respectively.Genetic and phenotypic correlations were estimated using multivariate analysis using the best fit model for the data set.

Model comparison
Spermatogenesis and sperm quality influencing factors such as sire birth year, birth type, birth season, location, body condition score, and dam parties were incorporated as fixed effects.Both sire nutrition and method of semen collection were not included as fixed effects because their influences were uniform for all sires.The significance test was examined after comparison of maximum log L with chi-square distribution at 95% confidence interval and the full and reduced model parameters difference as the degree of freedom.However, for the current dataset, comparison of AIC value was more appropriate than chi-square distribution.As a result, model 1 is the best-fit model for the dataset and is indicated in bold (Table 2).The model yielding the smallest AIC value fits the data best (Akaike, 1974).In the current dataset, both maternal genetic effect and maternal permanent environmental effect didn't significantly affect genetic parameters estimations.As a result, an only additive genetic effect was sufficient to evaluate semen characteristics for Bonga sheep.

Variance components and heritability estimation
The estimates of variance component and heritability for studied traits of semen characteristics are presented in Table 3.The variance components of the best-fit model (model 1) for each trait indicated that 0.03, 0.06, 0.11, 0.39, 3.28, and 0.52, of the total variation embraced of direct additive variances for semen volume, sperm mass motility, sperm concentration, total spermatozoa, six months body weight, and scrotum circumference, respectively (Table 3).The percentage share of additive effects from phenotypic value of listed traits were 20.0%, 13.6%, 12.6%, 9.8%, 37.1%, 10.9%, and 33.2%, respectively.
The narrow sense heritability of the studied traits for semen volume, sperm mass motility, sperm concentration, total spermatozoa, six months body weight, and scrotum circumference, are 0.23 ± 0.122, 0.13 ± 0.129, 0.13 ± 0.110, 0.09 ± 0.036, 0.37 ± 0.155, and 0.11 ± 0.162, respectively.This indicated that sperm mass motility, sperm concentration, total spermatozoa, and scrotum circumference traits have low heritability while volume of semen and sire selection age of six months weight have medium heritability.A trait that has low heritability indicated that major variation among individual is due to environmental effects and can't improve by selection.However, for medium heritability traits both genetic and environment have equal contributing factors to express the performance of the animal.

Genetic and phenotypic correlation
Correlations among traits were estimated using the best-fit model (model 1).Multivariate analysis revealed semen volume has negative and high genetic correlations with all studied traits except total spermatozoa (0.66 ± 0.213) (Table 4).The sperm mass motility trait is negatively correlated with its concentration (−0.34 ± 0.102) and total spermatozoa (−0.42 ± 0.411).The association between six months' body weight of sire with its sperm mass motility and concentration are    where SMWT = six months body weight; TSP = total spermatozoa; and SC = scrotum circumference.
positive and highly correlated (0.58 ± 0.301 and 0.45 ± 0.206), respectively.However, this selection weight has a negative correlation with semen volume and total spermatozoa production (Table 4).Scrotum circumference has a positive and high correlation with sires six months body weight, semen motility and concentration 0.83 ± 0.598, 0.54 ± 0.299, and 0.55 ± 0.144, respectively, but negative with semen volume and total spermatozoa (Table 4).

Discussion
The value accounted for 20.0%, 13.6%, 12.6%, 9.8%, 37.1%, and 10.9%, of the total phenotypic variance was from additive effect for studied traits of semen volume, sperm mass motility, sperm concentration, total spermatozoa, six months body weight, and scrotum circumference, from the best-fit model.This indicated that animal environments share a higher percentage to express the trait of interest.Both reproductive and fertility traits are mainly affected by their environment.Semen traits like ejaculated volume, mass motility, and concentration are crucial determinants of successful reproduction and then can influence the profitability of commercial sheep enterprises (Hodge et al., 2022).
The magnitude of heritability ranges among the six different models was from 0.07 ± 0.101 to 0.23 ± 0.122 for semen volume, 0.10 ± 0.012 to 0.31 ± 0.100 for sperm mass motility, 0.11 ± 0.210 to 0.32 ± 0.280 for sperm concentration, 0.02 ± 0.112 to 0.32 ± 0.255 for total spermatozoa, 0.23 ± 0.062 to 0.37 ± 0.155 for six months weight, and 0.05 ± 0.142 to 0.28 ± 0.090 for scrotum circumference.The heritability of semen characteristics and sire selection traits ranges by different model from low to medium.In support of the current finding, few studies explained that heritability of semen traits ranged between generally low to moderate (Hodge et al., 2022).Similarly, the heritability of volume, motility and concentration of semen range from 0.077 to 0.304 for Spanish dairy rams across five breeds (Rege et al., 2000).The heritability of six months weight from the best-fit model was 0.37 ± 0.155 and categorized under medium heritability.The implication of the heritability value for Bonga sheep was a possibility of further improvement for growth traits through selection.The current value of heritability for six months' weight was higher than the previous estimation of Bonga sheep, Doyogena sheep, Horro sheep, and Menz sheep of Ethiopia (Areb et al., 2021;Habtegiorgis et al., 2020;Haile et al., 2020).The heritability of semen volume was higher than Ethiopian highland sheep (Horro and Menz) (0.07 ± 0.22) and Hereford bull (0.09 ± 0.08) (Kealey et al., 2006;Rege et al., 2000) but lower for mass motility (Kealey et al., 2006;Rege et al., 2000).Heritability of semen volume was higher than Chinese Holstein bulls (0.15), related semen motility (0.12), and lower concentration (Yin et al., 2019).The heritability of scrotum circumference in the current study was low (0.11 ± 0.162).Contrary (Morris et al., 1992) explained that measure of testicular measurement was used as a selection criterion to improve the fertility of cattle and sheep due to its high heritability.The current heritability of the value of scrotum circumference and body weight of Bonga sheep was lower than Merino sheep 0.25 to 0.40 and 0.49 to 0.52, respectively (Duguma et al., 2002).Also, Barth (2007) reviewed that the heritability of scrotal circumference in young bulls is ~ 0.5, it responds well to selection and the Hereford bull (0.57 ± 0.09) (Kealey et al., 2006).
The wave motion of sperm mass motility is an indicator of semen quality and sire fertility (David et al., 2015) but is low heritability for Bonga sheep (0.13 ± 0.129).The low heritability of semen traits (motility and concentration) for Bonga sheep was in agreement with Australian sheep (Hodge et al., 2022).
The phenotypic associations of all studied traits are to be positive.Similarly, conception outcomes are positively correlated with gross motility and conception for sheep (David et al., 2015).Large and healthy scrotum circumference and estimated breeding value at six months body weight are the major selection criteria for breeding sire of Bonga sheep under CBBP.The current study revealed that negative association of semen volume with both six months' body weight and scrotum circumference which may have its own disadvantage by reducing the number of straws during artificial insemination of ewes.Also, this association may influence the profitability of seed stock production enterprises involved in the sale of semen doses used in artificial breeding for the future.Volume and concentration of semen influence the quantity of insemination doses produced from an ejaculate (Hodge et al., 2022).Similarly, the correlation between volume and concentration has been reported to vary between −0.71 and 0.02 for Latxa Cara Rubia and Churra rams (Pelayo et al., 2019).In support of the current research ejaculate volume was negatively correlated with percentages of live and motile sperm for Hereford bull (−0.09 and −0.38, respectively) and Latxa Cara Rubia Spanish dairy sheep (−0.37) (Kealey et al., 2006;Pelayo et al., 2019).Oppositely semen volume has positive and high correlation (0.92 and 0.91) with scrotum circumference for Holstein Frisian and Brahman bulls, respectively, and crossbreed Sahiwal (0.72) (Ha et al., 2012;Latif et al., 2009).Scrotum circumference has a positive and high correlation with six months' body weight, motility, and concentration.Similarly, scrotum circumference has a positive and high correlation with body weight (0.89) for the Kivircik ram lamb of Turkey (Elmaz et al., 2007), and Holstein Frisian and Brahman bull (0.72) (Ha et al., 2012).Also, bulls having bigger scrotum and earlier puberty produce better semen (Siddiqui et al., 2008).As a result, using scrotum circumference as a criterion for breeding sire selection may have a positively correlated response on semen qualities.Similarly, (Elmaz et al., 2007) explained that scrotum circumference can be used as a criterion for the early selection of ram lambs to be used in breeding at relatively young ages.Six months' body weight was positively correlated with sperm mass motility and concentration which may have a correlated response for the selection program because these traits are indicators of semen quality.The differences on both heritability and correlation may be due to different in breed, species, genetic background, type of models used for estimation of parameters, and the sample size for analysis.

Conclusion
Most of the studied semen characteristics traits have low heritability.Therefore, within-breed selection may not attain significant improvement but the management of animals such as feed, age, interval between semen collection may have the lion's share to get better performance of semen qualities.However, the breeding sire selection age of six months body weight and semen volume have moderate heritability.A positive association of both scrotum circumference and six months of body weight with sperm mass motility and concentration may have a correlated response for semen quality under the implementation of CBBP.Application of scrotum circumference as selection criteria in addition to other traits would have a correlated response with body weight.The current studied data on semen characteristics were collected from a single season that results unable to evaluate the seasonal variation of semen characteristics.Also, the research lacks a morphological defect (abnormality) test for spermatozoa.These shortages in the study will be further investigated in the future.
2), additive maternal (σ m 2 ), and maternal permanent environmental (σ c 2 ) of direct, maternal, residual, maternal permanent environment, and phenotypic respectively; σ am covariance between direct and maternal; maternal, and ratio of maternal permanent environmental variance to phenotypic variance; and r am : genetic correlation between direct and maternal.

Table 1 . Data structure for identified breeding sires of Bonga sheep under CBBP Items Values
Where: CBBP = community based breeding program; No. = number.

Table 2 . Best-fit model selection for the data set of breeding sires of Bonga sheep under CBBP
where CBBP = community based breeding program; Max Log L = maximum log likelihood; and AIC = Akaike information Criteria.