Bee drone brood is a beehive by-product with high hormonal activity used in natural medicine to treat male infertility. The aim of the study was to assess the effect of drone brood on stallion spermatozoa during a short-term incubation for its potential use in the equine semen extenders. Three different forms of fixed drone brood (frozen (FR), freeze-dried (FD), and dried extract (DE)) were used. Solutions of drone brood were compared in terms of testosterone, protein, total phenolic content, and antioxidant activity. The stallion semen was diluted with prepared drone brood solutions. The computer-assisted semen analysis (CASA) method was employed to evaluate the movement characteristics of the diluted ejaculate. To determine spermatozoa viability, the mitochondrial toxicity test (MTT) and Alamar Blue test were performed. In terms of testosterone content and antioxidant activity, a close likeness between FR and FD was found whereas DE\'s composition differed notably. FR had a positive effect mainly on progressive motility, but also on sperm distance and speed parameters after 2 and 3 h of incubation. On the contrary, FD and DE acted negatively, depending on increasing dose and time. For the first time, a positive dose-dependent effect of fixed drone brood on spermatozoa survival in vitro was demonstrated.

Probiotic and prebiotic effects on equine semen and gastrointestinal microbiome composition and sperm quality are unknown. This study aimed to evaluate the effects of pre-, pro- or synbiotic supplementation on fecal and semen microbiome composition and sperm quality parameters of stallions. This Latin square crossover trial involved four miniature pony stallions receiving control diet only, or addition of a pro-, pre- or synbiotic formulation. Full-length 16S rRNA gene amplicon sequencing was used to measure diversity of semen and fecal microbiomes. Total sperm count, total motility, progressive motility, DNA integrity, lipid peroxidation and mitochondrial oxidative stress, biomarkers of sperm quality, were measured after each intervention. A general linear model was employed to analyse and compare microbiome diversity measures and sperm quality data across four time points. Shannon\'s diversity index (alpha-diversity), and evenness of semen and gastrointestinal microbiomes were significantly different (p<0.001). A trend was observed for prebiotic effects on the diversity indices of the GI microbiome (p= 0.07). No effects of treatments were observed on either semen microbiome or sperm quality. Pre-, pro- and synbiotic supplements showed no negative effect on sperm quality parameters observed. This proof of concept provides preliminary data to inform future studies exploring the relationship between microbiomes and fertility.

A consistently high level of stallion fertility plays an economically important role in modern horse breeding. To better understand the factors affecting variation in stallion fertility, we have performed a statistical analysis study on some breeding factors: year of breeding, stud farm, age of the stallion, number of covered mares per stallion, reproduction methods, and age of the mare. This work was conducted on 94 purebred Arabian stallions in four different regions of Tunisia. The results showed an increase in the number of stallions during the study period, ranging from 11.33% in 2011 to 13.29% in 2018. Sidi Thabet\'s stud farm contained the highest number of purebred Arabian stallions. The majority of stallions were between 15 and 21 years old and had covered 1 to 20 mares; 95.19% of stallions were used in natural mating (Nat); 50.36% had low fertility, 17.69% had medium fertility, and 32.3% had excellent fertility according to fertility standards. Depending on the year and stud, there was a variation in fertility per cycle (FERPCE) and end-of-season fertility (FERPSE) of the stallions. The highest average FERPCE and FERPSE values were obtained using artificial insemination with fresh semen (AIF). Analysis of FERPCE and FERPSE showed that the model used in our study explained 40.21% of total variability observations for FERPCE and 42.1% for FERPSE. The used statistical model showed that the breeding year, the stud, the age of the stallion, the number of covered mares by stallions and the method of reproduction significantly affected both FERPCE and FERPSE (with p = 0.001). Low to moderate heritability estimations for FERPCE (hs2 = 0.08) and FERPSE (hes2 = 0.36) were obtained.

OBJECTIVE: The semen quality of stallions including sperm motility is an important target of selection as it has a high level of individual variability. However, effects of the molecular architecture of the genome on the mechanisms of sperm formation and their preservation after thawing have been poorly investigated. Here, we conducted a genome-wide association study (GWAS) for the sperm motility of cryopreserved semen in stallions of various breeds.
METHODS: Semen samples were collected from the stallions of 23 horse breeds. The following semen characteristics were examined: progressive motility (PM), progressive motility after freezing (FPM), and the difference between PM and FPM. The respective DNA samples from these stallions were genotyped using Axiom Equine Genotyping Array.
RESULTS: We performed a GWAS search for single nucleotide polymorphism (SNP) markers and potential genes related to motility properties of frozen-thawed semen in the stallions of various breeds. As a result of the GWAS analysis, two SNP markers, rs1141327473 and rs1149048772, were identified that were associated with preservation of the frozen-thawed stallion sperm motility, the relevant putative candidate genes being NME/NM23 family member 8 (NME8), olfactory receptor family 2 subfamily AP member 1 (OR2AP1), and olfactory receptor family 6 subfamily C member 4 (OR6C4). Potential implications of effects of these genes on sperm motility are herein discussed.
CONCLUSIONS: The GWAS results enabled us to localize novel SNPs and candidate genes for sperm motility in stallions. Implications of the study for horse breeding and genetics are a better understanding of genomic regions and candidate genes underlying stallion sperm quality, and improvement in horse reproduction and breeding techniques. The identified markers and genes for sperm cryotolerance and the respective genomic regions are promising candidates for further studying the biological processes in the formation and function of the stallion reproductive system.

A high degree of inbreeding has been reported to negatively impact semen quality in Friesian horses and Shetland ponies. Both breeds are characterized by a closed studbook, small population size, and high incidence of inbreeding. The Dutch Warmblood studbook (KWPN: Koninklijk Warmblood Paardenstamboek Nederland) is a much larger studbook with two distinct populations: the KWPN-Riding horses, managed as an \'open\' studbook, and the KWPN-Harness horses, representing a much smaller subpopulation within the KWPN breed and managed as an \'almost closed\' studbook. It was recently reported that the degree of inbreeding in KWPN-Harness horses has increased in recent decades due to the small gene pool; however, the degree of inbreeding is still lower than that of Friesian horses and Shetland ponies. We hypothesized that a high or rising degree of inbreeding might negatively impact semen quality. In the present study, we retrospectively compared semen quality parameters of stallions from four different breeds or types (Friesian Horses, Shetland Ponies, KWPN-Riding horses, and KWPN-Harness horses), each reported with different degrees of inbreeding. Semen concentration, and percentages of motile, morphologically normal and live spermatozoa, and the total number of morphologically normal, progressive motile spermatozoa per ejaculate (TNM) were analyzed for 2832 semen evaluations performed over a 15-year period. KWPN-Harness horses had a significantly lower sperm concentration, % motile spermatozoa and % live spermatozoa than KWPN-Riding horses but the % motile and % morphologically normal spermatozoa and TNM in both KWPN-Harness and KWPN-Riding horses were significantly higher than in Friesian horses and Shetland ponies. These results suggest a lower semen quality in KWPN-Harness than KWPN-Riding horses, potentially as a result of a higher coefficient of inbreeding. The negative trend observed in the KWPN-Harness horses may be a warning sign, and breeders or stud books should monitor the degree of inbreeding carefully to avoid a further reduction in semen quality, to the levels observed in Friesian horses and Shetland ponies.

UNASSIGNED: The Barb horse occupies a prominent place in the history, culture, and equestrian traditions of the Maghreb (Algeria, Morocco, and Tunisia). Although many studies on the breed standard and morphometry have been published, there are no studies on its fertility and reproductive potential. Thus, this work aimed to study the fertility of Barb, Arabian Purebred, and Thoroughbred horses in Algeria.
UNASSIGNED: A total of 168 stallions and 1202 mares at the Chaouchaoua Stud farm in Tiaret, Algeria, were included in the study. The reproductive performance during 1592 cycles over 10 consecutive mating seasons (2003-2012) was evaluated. Multivariate analysis with logistic regression was used to analyze the reproductive parameters such as the number of cycles operated, number of pregnant mares, pregnancy rate per cycle, seasonal pregnancy rate, and embryonic mortality rate, and to determine the influence of breed, stallion\'s age, and year of the study on reproduction.
UNASSIGNED: Statistical analysis showed that stallion breed was a significant influencing factor for the number of pregnant barren mares (Odds ratio [OR]=1.72; p=0.03; 95% confidence interval [CI]=1.05, 2.84) seasonal pregnancy rate (OR=1.40; p<0.001; 95% CI=1.29, 1.53). Additionally, the reproductive performance of the Barb stallion was superior to that of the Thoroughbred stallion. With regard to the significant influence of stallion\'s age of >5 years on the number of pregnant foaling mares and seasonal pregnancy rate, significant interactions were observed between the stallion\'s breed and age, and the pregnancy rate per cycle: in the Barb breed, the pregnancy rate per cycle increased with the age of the stallion, while in the Thoroughbred, it decreased with age. Moreover, a significant effect of the year of the study on the pregnancy rate per cycle and number of pregnant foaling mares was observed. In contrast, the number of cycles and embryonic mortality rate were not influenced by the breed and age of the stallion, or the year of the study.
UNASSIGNED: The Algerian Barb horse attained a similar level of fertility compared with that of the Arabian Purebred and Thoroughbred stallions depending on its age and reproductive performance.