The analysis of runs of homozygosity (ROH), using high throughput genomic data, has become a valuable and frequently used methodology to characterize the genomic and inbreeding variation of livestock and wildlife animal populations. However, this methodology has been scarcely used in highly inbred domestic animals. Here, we analyzed and characterized the occurrence of ROH fragments in highly inbred (HI; average pedigree-based inbreeding coefficient FPED = 0.164; 0.103 to 0.306) and outbred Retinta bulls (LI; average FPED = 0.008; 0 to 0.025). We studied the length of the fragments, their abundance, and genome distribution using high-density microarray data. The number of ROH was significantly higher in the HI group, especially for long fragments (>8Mb). In the LI group, the number of ROH continuously decreased with fragment length. Genome-wide distribution of ROH was highly variable between samples. Some chromosomes presented a larger number of fragments (BTA1, BTA19, BTA29), others had longer fragments (BTA4, BTA12, BTA17), while other ones showed an increased ROH accumulation over specific loci (BTA2, BTA7, BTA23, BTA29). Similar differences were observed in the analysis of 12 individuals produced by a similar inbred event (FPED3 = 0.125). The correlation between the fraction of the genome covered by ROH (FROH) and FPED was high (0.79), suggesting that ROH-based estimations are indicative of inbreeding levels. On the other hand, the correlation between FPED and the microsatellite-based inbreeding coefficient (FMIC) was only moderate (r = 0.44), suggesting that STR-based inbreeding estimations should be avoided. Similarly, we found a very low correlation (r = -0.0132) between recombination rate and ROH abundance across the genome. Finally, we performed functional annotation analyses of genome regions with significantly enriched ROH abundance. Results revealed gene clusters related to pregnancy-associated proteins and immune reaction. The same analysis performed for regions enriched with recently formed ROH (> 8 Mb) showed gene clusters related to flagellum assembly. In both cases, the processes were related to male and female reproductive functions, which may partially explain the reduced fertility associated with inbred populations.

The Blanca Serrana goat is selected for meat production and usually raised in an extensive farm system. The meat goat industry is getting bigger in Spain, evolving to more intensive farming systems. The negative influence of stress produced by daily management on animal welfare is even bigger in these animals as they are not used to getting so close to humans. Eye temperature has recently appeared as an appropriate and noninvasive tool for welfare assessment in cattle, but no previous studies have been developed in goats. Thus, the main aim of this pilot study was to test eye temperature as a noninvasive tool to explore stress levels associated with a semi-intensive farming system for meat goats in comparison with the standard measurements of stress.For that, 24 Blanca Serrana goats were used. Heart rate (HR), respiratory rate (RR) and eye temperature (ET), assessed with infrared thermography samples, were collected just before and just after a stressful situation created to check how the routine management of semi-intensive farming systems affected this species. A factorial ANOVA, least square means and Scheffé post hoc comparison analyses found statistically significant differences due to the stress test moment for RR (p<0.05) and ET (p<0.001) with higher values shown after the stress test than before it. Differences due to age were found just for HR (p<0.05) and RR (p<0.01) stress parameters, with kids showing higher results than adults. Pearson correlations between HR, RR and ET parameters showed a medium–high positive correlation of 0.56 between RR and ET.Thus, ET appears as an appropriate and noninvasive tool to explore stress levels associated with a semi-intensive farming system for meat goats.

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Inbreeding depression, the genetic condition caused by mating related individuals, is particularly important in several cattle breeds with limited effective populations. This condition is often associated with decreases in performance, including fertility. Furthermore, sperm head morphometry was associated with fertility in several species. To our knowledge, the influence of inbreeding on sperm morphometry has not been widely reported in cattle. In this study, a Sperm Class Analyzer (SCA™, Microptic S.L., Barcelona, Spain) was used to characterise 7 sperm morphometry parameters in 59 Retinta bulls, including sperm head length, width, perimeter, ellipticity, elongation, regularity and rugosity. Two replicates of at least 100 sperm heads, from 2 frozen semen samples, were assessed per individual (n=200 sperm per bull). Additionally, all individuals were genotyped with the Axiom Bos1 high density SNP Array (Affymetrix, Santa Clara, CA, USA). The molecular-based inbreeding factor (FROH; mean 12.5%, range 1.75 to 33.0) had very low correlations with all sperm morphometry parameters. On average, sperm heads from bulls with higher FROH had a smaller area, but variability was high. Correlations between inbreeding and sperm shape were low and negative, length (r=−0.1449; P&lt;0.01), width (r=−0.2494; P&lt;0.01), and rugosity (r=−0.1086; P&gt;0.01) being the most informative. Whole-genome association study (GWAS) analysis, performed using molecular inbreeding as co-factor, revealed 8 SNP, located on chromosomes BTA2, BTA5, BTA7, and BTA11, significantly associated with sperm head regularity and rugosity. Genomic analysis revealed that genes SLF1 and TMEFF2 are located close to enriched SNP. Gene SLF1 (SMC5-SMC6 complex localization factor 1) is associated with the regulation of protein complex and cytoskeleton assembly, whereas TMEFF2 (transmembrane protein with EGF like and 2 follistatin like domains 2) is associated with integral components of cell membrane and cell spreading and development. Therefore, we inferred that SLF1 and TMEFF2 may be involved in variations of sperm head shape. In this preliminary study, there was evidence of a potential influence of inbreeding on sperm morphometry in a beef cattle breed. However, additional studies, ideally including more individuals and additional breeds, are necessary to determine the validity of this potential association.

The effect of inbreeding depression on sperm motility is well documented, but its influence on sperm morphometry has been scarcely examined to date. Here, we combined the use of computer‐assisted sperm morphometry analysis (CASMA) with a SNP‐based genomic approach to determine and characterize the effect of inbreeding on the sperm shape of a highly inbred cattle population. We determined seven morphometric parameters on frozen‐thawed sperm samples of 57 Retinta bulls: length (L, µm), width (W, µm), area (A, µm2), perimeter (P, µm), ellipticity (ELI; L/W), elongation (L−W)/(L + W) and perimeter‐to‐area shape factor (p2a; P2/4 × π × A). The comparison of highly inbred (HI) and lowly inbreed (LI) individuals based on runs of homozygosity (ROH) inbreeding values (F ROH) showed no differences between groups. An additional two‐step unsupervised sperm subpopulation analysis based on morphometric parameters showed significant differences in the abundance of different sperm subpopulations between groups (p < 0.05). This analysis revealed that HI bulls harbored a higher percentage of narrow‐head sperm as opposed to the higher percentage of large‐ and round‐headed sperm detected in LI. A further genomic characterization revealed 23 regions differentially affected by inbreeding in both groups, detecting six genes (SPAG6, ARMC3, PARK7, VAMP3, DYNLRB2, and PHF7) previously related to different spermatogenesis‐associated processes.

Reproductive performance is one of the key factors in livestock production. It is well known that reproductive traits are influenced by several genetic factors, such as the increase of individual inbreeding levels, which are associated with changes in sperm motility and shape in several species. In horses, the increase in inbreeding is a common problem because of the reduction in effective population size and the increase in selection intensity observed in several breeds. However, studies assessing the effect of high levels of inbreeding on the sperm quality of stallions are scarce. In the present study, we aimed to determine the effect of increased inbreeding levels and age on the sperm motility patterns of Purebred Spanish horses (PRE). We performed kinetic characterisation of 557 sperm samples of 82 PRE stallions aged between 3 and16 years, using computer-assisted sperm analysis (Androvision™, Minitube). We evaluated 5 parameters in 6 different fields per sample: curved line velocity (VCL, µm/s), velocity average path (VAP, µm/s), velocity straight line (VSL, µm/s), amplitude of lateral head displacement (ALH, µm), and beat-cross frequency (BCF, Hz). We determined the pedigree-based inbreeding coefficient (Fped) based on ∼300,000 PRE pedigree records to evaluate the inbreeding effect. Individuals were separated into 2 groups: highly inbred (n=339) and lowly inbred (n=218) according to an F value of 12.5%. Differences between groups were analysed using a generalized linear model. The analysis did not show significant differences (P&gt;0.05) in the variables analysed with respect to the age of stallions. However, VAP, VCL, and AHL were lower in highly inbred than in lowly inbred animals (P&lt;0.05), suggesting less velocity and amplitude of head displacement. In the case of BCF, no significant differences (P&gt;0.05) were observed between the two study groups. In conclusion, age did not affect sperm quality parameters in the age group of stallions analysed. In addition, we demonstrated that high inbreeding coefficient reduced the mean velocity and trajectory pattern of spermatozoa in PRE.

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