Equine Breeding Research Articles

Prevalence of Taylorella equigenitalis in Icelandic mares and geldings in Southern Germany and Austria

Contagious Equine Metritis (CEM) caused by the bacterium Taylorella equigenitalis (T. equigenitalis), is a venereal infection of equids which is of international concern to the equine breeding industry. A recent study showed a high prevalence of T. equigenitalis in Icelandic stallions when compared to stallions of other breeds also using for natural breeding. Consequently, the objectives of the present study were to investigate the prevalence of T. equigenitalis in Icelandic mares and geldings and to determine factors associated with a T. equigenitalis-positive qPCR result. In total, 361 Icelandic horses located in Southern Germany and Austria were tested for T. equigenitalis using a qPCR assay. An overall prevalence of 14.4% was detected. Positive qPCR results were found in 2.2% (3/134) of brood mares, 9.0% (11/122) of maiden mares and in 36.2% (38/105) of geldings. The odds for a T. equigenitalis-positive qPCR result were significantly lower in both brood (OR=40.1, 95% CI: 8.38-192, P<0.001) and maiden mares (OR=9.51, 95% CI: 3.26-25.7, P<0.001) when compared to geldings. Advancing age was not associated with higher odds for a T. equigenitalis-positive qPCR result (OR=0.98, 95% CI: 0.94-1.03, P=0.51). However, horses of the younger age group showed significantly lower Ct values compared to horses of the older age group (P=0.04). Furthermore, geldings showed significantly lower Ct values than brood (P<0.03) and maiden mares (P<0.001). This study showed a significantly higher prevalence of T. equigenitalis in Icelandic geldings compared to Icelandic mares. Icelandic geldings might therefore represent a reservoir for T. equigenitalis.

Unveiling Genetic Potential for Equine Meat Production: A Bioinformatics Approach.

In view of the predicted significant increase in global meat production, alternative sources such as horsemeat are becoming increasingly important due to their lower environmental impact and high nutritional value. This study aimed to identify SNP markers on the GeneSeek® Genomic Profiler™ Equine (Neogen, Lansing, MI, USA) that are important for horsemeat production traits. First, orthologous genes related to meat yield in cattle and common genes between horses and cattle within QTLs for body size and weight were identified. Markers for these genes were then evaluated based on predicted variant consequences, GERP scores, and positions within constrained elements and orthologous regulatory regions in pigs. A total of 268 markers in 57 genes related to meat production were analyzed. This resulted in 27 prioritized SNP markers in 22 genes, including notable markers in LCORL, LASP1, IGF1R, and MSTN. These results will benefit smallholder farmers by providing genetic insights for selective breeding that could improve meat yield. This study also supports future large-scale genetic analyses such as GWAS and Genomic Best Linear Unbiased Prediction (GBLUP). The results of this study may be helpful in improving the accuracy of genomic breeding values. However, limitations include reliance on bioinformatics without experimental validation. Future research can validate these markers and consider a wider range of traits to ensure accuracy in equine breeding.

An Overview of Infectious and Non-Infectious Causes of Pregnancy Losses in Equine.

Equine breeding plays an essential role in the local economic development of many countries, and it has experienced rapid growth in China in recent years. However, the equine industry, particularly large-scale donkey farms, faces a significant challenge with pregnancy losses. Unfortunately, there is a lack of systematic research on abortion during equine breeding. Several causes, both infectious and non-infectious, of pregnancy losses have been documented in equines. The infectious causes are viruses, bacteria, parasites, and fungi. Non-infectious causes may include long transportation, ingestion of mycotoxins, hormonal disturbances, twinning, placentitis, umbilical length and torsion, etc. In current review, we discuss the transmission routes, diagnostic methods, and control measures for these infectious agents. Early detection of the cause and appropriate management are crucial in preventing pregnancy loss in equine practice. This review aims to provide a comprehensive understanding of the potential causes of abortion in equines, including infectious agents and non-infectious factors. It emphasizes the importance of continued research and effective control measures to address this significant challenge in the equine industry.

Effect of bicarbonate and polyvinyl alcohol on in vitro capacitation and fertilization ability of cryopreserved equine spermatozoa.

Factors contributing to the limited success of in vitro fertilization in horses remain to be studied. In this work, we elucidated the effect of different essential capacitation media components, bicarbonate, and bovine serum albumin or polyvinyl-alcohol, and the incubation microenvironment on sperm parameters associated with capacitation, acrosome reaction, and their ability to activate oocytes via heterologous intracytoplasmic spermatozoa injection in equine cryopreserved spermatozoa. Frozen-thawed spermatozoa underwent incubation at different time intervals in either Tyrode's albumin lactate pyruvate medium (non-capacitating; NC) or Tyrode's albumin lactate pyruvate supplemented with bicarbonate, bicarbonate and polyvinyl-alcohol, bicarbonate and bovine serum albumin, polyvinyl-alcohol and bovine serum albumin alone. Protein kinase A-phosphorylated substrates and tyrosine phosphorylation levels, sperm motility, and acrosome reaction percentages were evaluated. After determining the best condition media (capacitating; CAP), heterologous intracytoplasmic spermatozoa injection on pig oocytes was performed and the phospholipase C zeta sperm localization pattern was evaluated. Incubation of frozen-thawed equine spermatozoa with bicarbonate and polyvinyl-alcohol in atmospheric air for 45min induced an increase in protein kinase A-phosphorylated substrates and tyrosine phosphorylation levels compared to NC condition. Sperm incubation in bicarbonate and polyvinyl-alcohol medium showed an increase in total motility and progressive motility with respect to NC (p ≤ 0.05). Interestingly, three parameters associated with sperm hyperactivation were modulated under bicarbonate and polyvinyl-alcohol conditions. The kinematic parameters curvilinear velocity and amplitude of lateral head displacement significantly increased, while straightness significantly diminished (curvilinear velocity: bicarbonate and polyvinyl-alcohol=120.9±2.9 vs. NC=76.91±6.9µm/s) (amplitude of lateral head displacement: bicarbonate and polyvinyl-alcohol=1.15±0.02 vs. NC=0.77±0.03µm) (straightness: bicarbonate and polyvinyl-alcohol=0.76±0.01 vs. NC=0.87±0.02) (p ≤ 0.05). Moreover, the spontaneous acrosome reaction significantly increased in spermatozoa incubated in this condition. Finally, bicarbonate and polyvinyl-alcohol medium was established as CAP medium. Although no differences were found in phospholipase C zeta localization pattern in spermatozoa incubated under CAP, equine spermatozoa pre-incubated in CAP condition for 45min showed higher fertilization rates when injected into matured pig oocytes (NC: 47.6% vs. CAP 76.5%; p ≤ 0.05). These findings underscore the importance of bicarbonate and polyvinyl-alcohol in supporting critical events associated with in vitro sperm capacitation in the horse, resulting in higher oocyte activation percentages following heterologous intracytoplasmic spermatozoa injection. This protocol could have an impact on reproductive efficiency in the equine breeding industry.

A case of Brucellosis with neurological and dermatological findings in a thoroughbred Arabian horse

Brucellosis, a zoonotic infection, exhibits varying prevalence across different geographical regions, with a notable incidence in Mediterranean countries, contributing to considerable economic losses. This study focuses on an 11-year-old purebred male Arabian horse, presenting symptoms including weight loss, weakness, environmental indifference, joint swelling, and diverse dermatological manifestations despite sufficient feed intake. A pivotal clinical observation from anamnesis and examination is the animal's inclination to lean its head against walls or fixed objects. In conjunction with hematological and biochemical analyses, the Brucella Rose Bengal plate test (RBPT) and serum agglutination test (SAT) were conducted, revealing seropositivity for brucellosis in the patient. Consequently, it is imperative for professionals in the equine breeding sector, particularly clinical veterinarians, to adhere to biosafety protocols concerning brucellosis cases exhibiting atypical clinical symptoms in horses. Proper diagnostic methods should be employed to assess suspicious cases, underscoring the significance of safeguarding both public and animal health.

A combination of biomarkers for predicting stallion sperm fertility

Equine breeding would benefit greatly from reliable biomarkers of stallion or ejaculate fertility. The aim of the study was to investigate how several in vitro sperm characteristics correlate with fertility after artificial insemination, to explore the potential to build a fertility prediction model for stallions. Cooled insemination doses (3–5 per stallion) were obtained from various studs. Sperm membrane integrity, acrosome integrity, chromatin integrity, mitochondrial membrane potential, and reactive oxygen species production were evaluated by flow cytometry 24–30 h after semen collection, and sperm motility was assessed by computer aided sperm analysis. Calcein violet was used to differentiate viable spermatozoa. Per season pregnancy rates for these stallions were available the following year. Positive correlations were found between pregnancy rate and straightness (r = 0.43, p ≤ 0.001), as well as pregnancy rate and the proportion of living hydrogen peroxide positive spermatozoa (r = 0.32, p ≤ 0.05). There were negative correlations between pregnancy rate and amplitude of lateral head displacement (r = -0.26, p ≤ 0.05), and between pregnancy rate and the mean fluorescence of dead superoxide positive spermatozoa (r = -0.46, p < 0.001). Principal component analysis indicated that motility, membrane integrity, DNA fragmentation, and reactive oxygen species production were associated with pregnancy rate. Therefore, a combination of these factors could be used as a biomarker of fertility when assessing ejaculates. However, data from more individuals would be required to construct a model for fertility prediction.

Single-Cell Transcriptome Sequencing Reveals Molecular Expression Differences and Marker Genes in Testes during the Sexual Maturation of Mongolian Horses.

This study aimed to investigate differences in testicular tissue morphology, gene expression, and marker genes between sexually immature (1-year-old) and sexually mature (10-year-old) Mongolian horses. The purposes of our research were to provide insights into the reproductive physiology of male Mongolian horses and to identify potential markers for sexual maturity. The methods we applied included the transcriptomic profiling of testicular cells using single-cell sequencing techniques. Our results revealed significant differences in tissue morphology and gene expression patterns between the two age groups. Specifically, 25 cell clusters and 10 cell types were identified, including spermatogonial and somatic cells. Differential gene expression analysis highlighted distinct patterns related to cellular infrastructure in sexually immature horses and spermatogenesis in sexually mature horses. Marker genes specific to each stage were also identified, including APOA1, AMH, TAC3, INHA, SPARC, and SOX9 for the sexually immature stage, and PRM1, PRM2, LOC100051500, PRSS37, HMGB4, and H1-9 for the sexually mature stage. These findings contribute to a deeper understanding of testicular development and spermatogenesis in Mongolian horses and have potential applications in equine reproductive biology and breeding programs. In conclusion, this study provides valuable insights into the molecular mechanisms underlying sexual maturity in Mongolian horses.

Post-cooling semen processing and sperm re-suspension as an alternative method to circumvent poor semen cooling in stallions.

Artificial insemination with cooled-shipped semen is the primary method used in the equine breeding industry; yet, sperm quality and fertility can be suboptimal for some stallions when standard techniques are used. Therefore, there is a critical need to develop alternative approaches for these stallions. To assess sperm quality parameters and fertility of cooled-stored stallion semen processed by SpermFilter® or centrifugation and resuspended in three extenders. Controlled and field study. In Experiment 1, semen was collected from 21 stallions classified as having good ('Good-coolers', n = 8) or poor ('Bad-coolers', n = 13) semen cooling. The semen was extended at 30 million spermatozoa/mL in a skimmed milk-based (SM) diluent, and refrigerated for 24 h. Then, the cooled-stored semen was processed through SpermFilter® or centrifugation, and the resulting sperm pellets were resuspended in SM, SM containing pentoxifylline (SM-P), or an egg yolk-based (EY) extender. Unprocessed cooled-stored semen served as control. Sperm motility parameters, plasma membrane integrity (PMI), and mitochondrial membrane potential (HMMP) were assessed in cooled-semen pre- and post-processing. Experiment 2, cooled semen from 9 stallions classified as Bad-coolers was used to inseminate 18 embryo donor mares at 66 cycles (Unprocessed, n = 22; SpermFilter®/SM-P, n = 16; or SpermFilter®/EY, n = 28). Data were analysed with a mixed model and Tukey's as posthoc, and logistic regression. Processed semen resuspended in EY had superior sperm motility compared to unprocessed, SM and SM-P (p < 0.0001). Semen processed by SpermFilter® resuspended in SM-P was similar to EY (p > 0.05). Pellet resuspension with EY and SM-P improved the HMMP of Bad-cooler stallions (p = 0.0010). Semen processed by SpermFilter® had superior PMI to centrifuged semen (p < 0.0001). Mares inseminated with SpermFilter®/SM-P (50%, 8/16) or SpermFilter®/-EY (68%, 9/28) had higher pregnancy rates than mares bred with unprocessed semen (14%, 3/22) (p < 0.001). Low number of mares in the fertility trial. Sperm quality and fertility of Bad-cooler stallions can be enhanced by SpermFilter® and pellet resuspension with either EY or SM-P.

Factors affecting the efficiency of equine embryo transfer (EET) in polo mares under subtropical conditions of Pakistan.

Equine embryo transfer (EET) is a prominent technology in the equine breeding industry, and its efficacy is affected by a number of factors. The current study aimed to determine the effects of the breed of donor/recipient mares, estrus/ovulation induction treatment, cooled transportation of embryos, and synchrony between donor and recipient mares on the efficiency of the EET under subtropical conditions of Pakistan. A total of eighty-four (n = 84) Polo-playing donor mares (Argentino-polo = 41 and Anglo-Arab = 43) and seventy (n = 70) recipient mares (light breed = 26 and heavy breed = 44) were used for EET. The donor mares exhibiting natural estrus (n = 28) were detected by teaser a stallion, and corpus luteum (CL) having mares (n = 56) were treated with prostaglandin (150 μg of Cloprostenol) for estrus induction. The mares' follicular growth was monitored through ultrasonography until the dominant follicle's size reached 35 mm or more with a moderate to obvious uterine edema score. Afterward, the mares were treated either with GnRH, i.e., 50 μg of Lecirelin acetate (n = 41) or Ovusyn, i.e., 1500 IU hCG (n = 43). Insemination with chilled semen was performed 24 hours later. The embryos were collected non-surgically, 7 or 8 days after ovulation, from the donor mares. The collected embryos were transferred into the well-synchronized recipient mares as fresh (n = 44) or chilled (n = 26) embryos. The pregnancy after ET was checked through ultrasonography. Statistical analysis revealed that the embryo recovery rate (ERR) remained significantly higher (P<0.05) for the Prostaglandin (PG) treated group of donors as compared to the natural heat group of donors. The breed of donor mares, type of ovulatory treatment given, and day of embryo collection did not significantly (P>0.05) affect the ERR. There was no significant effect of the type (fresh vs chilled), classification, and stage of development of embryo on pregnancy outcomes (P>0.05). ET pregnancy rate was significantly affected by the breed of recipient mares and ovulation synchrony between donor and recipient mares (P<0.05). In conclusion, under the subtropical conditions of Pakistan, PG-based estrus induction of donor mares, breed of recipient mares, and ovulation synchrony between the donor and recipient mares had a substantial effect on the efficiency of EET.

Can Nicotinamide Adenine Dinucleotide (NAD+) and Sirtuins Be Harnessed to Improve Mare Fertility?

Years of sire and dam selection based on their pedigree and athletic performance has resulted in a reduction in the reproductive capability of horses. Mare age is considered a major barrier to equine reproduction largely due to an increase in the age at which mares are typically bred following the end of their racing career. Nicotinamide adenine dinucleotide (NAD+) and its involvement in the activation of Sirtuins in fertility are an emerging field of study, with the role of NAD+ in oocyte maturation and embryo development becoming increasingly apparent. While assisted reproductive technologies in equine breeding programs are in their infancy compared to other livestock species such as cattle, there is much more to be learnt, from oocyte maturation to early embryo development and beyond in the mare, which are difficult to study given the complexities associated with mare fertility research. This review examines what is already known about the role of NAD+ and Sirtuins in fertility and discusses how NAD+-elevating agents may be used to activate Sirtuin proteins to improve equine breeding and embryo production programs both in vivo and in vitro.

Monthly and Pregnancy-Related Concentration of Cu and Zn in Serum of Mares in an Equine Breeding Herd

Copper (Cu) and zinc (Zn) are trace minerals with multiple biological functions, playing roles in fetal development and immune regulation. Despite their known significance, research on Cu and Zn administration and supplementation for pregnant mares is insufficient. This study aims to evaluate Cu and Zn serum concentrations monthly throughout the year and through pregnancy in Polish Konik mares. The study was conducted in 2020 on 36 mares from the Polish state stud farm, with 24 mares in the pregnant group and 12 mares in the non-pregnant group. Monthly blood samples were collected, and serum Cu and Zn concentrations were measured. The total Cu concentration in the serum of all mares was higher (p &lt; 0.0001) in August and September (13.98 ± 3.00 μmol/L) than in May, June, July, and November (11.04 ± 2.74 μmol/L). Similarly, the total Zn concentration was higher (p &lt; 0.0001) in August, September, October, November, and December (19.80 ± 9.72 μmol/L) than in May, June, and July (14.50 ± 6.94 μmol/L). No evidence of a linear relation between Cu and Zn concentrations was demonstrated (slopes difference p &lt; 0.001). No differences in Cu (p &gt; 0.05) or Zn (p &gt; 0.05) concentrations were found between pregnant (Cu: 12.08 ± 3.01 μmol/L; Zn: 18.02 ± 9.03 μmol/L) and non-pregnant (Cu: 11.23 ± 2.51 μmol/L; Zn: 18.59 ± 10.37 μmol/L) mares in any of the examined months of the year. In conclusion, the month of the year, rather than pregnancy, affects serum Cu and Zn concentrations.

Effect of Sampling Method on Detection of the Equine Uterine Microbiome during Estrus.

Bacterial endometritis is among the most common causes of subfertility in mares. It has a major economic impact on the equine breeding industry. The sensitivity of detecting uterine microbes using culture-based methods, irrespective of the sample collection method, double-guarded endometrial swab, endometrial biopsy, or uterine low-volume lavage (LVL), is low. Therefore, equine bacterial endometritis often goes undiagnosed. Sixteen individual mares were enrolled, and an endometrial sample was obtained using each method from all mares. After trimming, quality control and decontamination, 3824 amplicon sequence variants were detected in the dataset. We found using 16S rRNA sequencing that the equine uterus harbors a distinct resident microbiome during estrus. All three sampling methods used yielded similar results in composition as well as relative abundance at phyla (Proteobacteria, Firmicutes, and Bacteroidota) and genus (Klebsiella, Mycoplasma, and Aeromonas) levels. A significant difference was found in alpha diversity (Chao1) between LVL and endometrial biopsy, suggesting that LVL is superior at detecting the low-abundant (rare) taxa. These new data could pave the way for innovative treatment methods for endometrial disease and subfertility in mares. This, in turn, could lead to more judicious antimicrobial use in the equine breeding industry.

Good Mothers or Lascivious Females? The Perception of Mares in the Context of Equine Husbandry and Breeding in Medieval France (1200-1500)

Though mares were essential to the breeding of medieval warhorses, they are elusive in written and iconographic sources dominated by male horses. When they appear, their portrayal is ambiguous: thirteenth-century encyclopaedists describe them both as an ideal representation of motherhood and as lascivious beings. This may have influenced the perception of mares in the context of equine husbandry. This paper compares descriptions in thirteenth-century encyclopaedias to French versions of Rufus’s De medicina equorum (1250), and to its 1456 reinterpretation by Guillaume de Villiers. The Roman d’Alexandre (1180) by Alexandre de Paris, and the financial records of Charles VI (1368-1422) are used to understand the complex cultural perception of mares. Despite negative sexual undertones, mares were portrayed as strong and independent, eliciting wariness and admiration.

New genomic insights into the conformation of Lipizzan horses

Conformation traits are important selection criteria in equine breeding, as they describe the exterior aspects of the horse (height, joint angles, shape). However, the genetic architecture of conformation is not well understood, as data of these traits mainly consist of subjective evaluation scores. Here, we performed genome-wide association studies on two-dimensional shape data of Lipizzan horses. Based on this data, we identified significant quantitative trait loci (QTL) associated with cresty neck on equine chromosome (ECA)16 within the MAGI1 gene, and with type, hereby differentiating heavy from light horses on ECA5 within the POU2F1 gene. Both genes were previously described to affect growth, muscling and fatty deposits in sheep, cattle and pigs. Furthermore, we pin-pointed another suggestive QTL on ECA21, near the PTGER4 gene, associated with human ankylosing spondylitis, for shape differences in the back and pelvis (roach back vs sway back). Further differences in the shape of the back and abdomen were suggestively associated with the RYR1 gene, involved in core muscle weakness in humans. Therefore, we demonstrated that horse shape space data enhance the genomic investigations of horse conformation.

Comparison of the efficacy of Lecirelin and hCG for ovulation induction in mares

Ovulating agents are very useful drugs in equine breeding, hCG being the first intention molecule, whereas GnRH agonists are especially interesting for any mare immunized against hCG, like repeat breeders and embryo donor mares. In Europe, no long acting GnRH agonist is available in veterinary practice. Saline solutions of buserelin or triptorelin, which is only available for human treatments in Europe are commonly used for equine ovulation induction (Dordas-Perpinya et al. Reprod. Dom. Anim. 2020; 55:374-383). An alternative, Lecirelin which is licensed in cattle and rabbits, has been evaluated in donkeys (Carluccio et al. Therio. 2007;68:914-919). The aim of our study was to compare the efficacy of lecirelin with hCG in horses. Forty mares underwent ovulation induction on four consecutive cycles, two cycles with hCG and two cycles with lecirelin, including one spontaneous estrus and one following chemical luteolysis. Mares were examined on a daily basis to check ovarian follicles diameter, uterine edema, and cervical softening. Once they presented a preovulatory follicle over 35 mm, and uterine edema (>=2) (Mc Kinnon. Equine diagnostic ultrasonography. 1998; 79-102) and a soft cervix, they were randomly injected with saline solution containing 200 µg of Lecirelin or 1500 IU of hCG intravenously. After induction, they were scanned every 12 hours until ovulation. Mares were inseminatedwith a mix of fresh semen of two fertile stallions 20 hours after induction on the spontaneous cycles. Pregnancy diagnosis was performed at day 12 and day 14. As each mare is her own control, statistical analysis was performed by χ², Fisher's test and Student's test for matched data. Follicle size at injection did not differ significantly between the two induction treatments (p>0,05), with or without distinction of spontaneous and induced cycles. Rates of ovulation in the 24-48 hours post-injection did not significantly differ between lecirelin (71%) and hCG (83%) on estrus following chemical luteolysis (p>0,05) or spontaneous estrus (43% for lecirelin and 68% for hCG) (p>0,05). We observed significantly more spontaneous ovulations occurring within the first 24 hours after induction with hCG (27% on spontaneous estrus, and 15% after chemical luteolysis) than with lecirelin (5% on spontaneous estrus, 7% after chemical luteolysis) (p < 0,05). Pregnancy rates were not significantly different following induction by lecirelin (67%) or hCG (57%) (p>0,05). These results highlight the usefulness of saline solution of lecirelin licensed in Europe for bovine and rabbit for induction and synchronization of ovulation for mares with control cycles. Lecirelin may be particularly suitable for embryo donor mares with a reduced hCG response.

US Doppler in the monitoring of hemodynamic alterations of the supplementary corpus luteum in cyclic mares

For the success of an equine breeding program, follow-up by a veterinarian is essential. In early pregnancy, luteinized follicles develop into an accessory or supplementary corpus luteum (SCL) which, like the primary corpus luteum, produces progesterone and contributes to the maintenance of early pregnancy (Hannan et al. Domestic Animal Endocrinology. 2020; 72:106424). If SCLs are insufficient, progesterone supplementation may be required but research data in this field are limited. Hence it is important to monitor the hemodynamic changes of the SCLs using Doppler ultrasonography (US Doppler). The aim of this study was to follow the onset and development of SCLs, evaluate their degrees of vascularization, subjectively (determining vascularity) and objectively (comparing the number and intensity of pixels). Crossbred mares (n=15) were inseminated, then SCLs monitored in Rio de Janeiro, Brazil. There was an average of 2.8 SCL/mare due to the ovulation and development of SCLs and differences in vascular perfusionwere seen between days 35 and 70 of gestation. There was a correlation (P<0.001) between SCL vascularization and the number and intensity of pixels, with the greatest increase occurring between days 70 and 119 of pregnancy. This corresponds with the peak production of progesterone by these structures. From day 120, a decrease in the vascular perfusion of the SCLs was seen, and at 140 days of gestation, the few SCLs that remained were in regression. This is logical because by this stage of gestation, progesterone is mainly of placental origin. Thus, US Doppler evaluation of SCLs in early pregnancy may assist in the decision whether to administer supplementary progesterone and for how long or not at all. Future research that may evaluate the contribution of equine chorionic gonadotropin to the formation of SCLs, its effect on the plasma concentration of progesterone as well as any requirement for progesterone supplementation at all is required.

The effect of sex-sorting on stallion semen quality

Spermatozoa retain fertilizing potential after sorting, but separation into X and Y populations has deleterious effects on the cell (Samper JC et al. Clinical Theriogenology 2022). The effect of sex-sorting (SS) on stallion sperm was evaluated in single ejaculates from 32 fertile, sexually active stallions. Based on previous work (Samper et al 2022) Botu-Gold was selected to dilute 2 billion cells at 1:3-1:4 ratio. Extender was supplemented with the DNA dye Hoechst 33342 and cooled at 8 ̊C for 18-24 h. Semen was warmed to 20 ̊C, and centrifuged through EquiPure at 200g for 30 min. The pellet was resuspended at 250-300 million/mL using ST Equiship, ST Genetics Texas) and transported at ∼20°C to the sorting facility. Sperm were SS (SexedULTRA™ Genesis III flow cytometer). Purity gate was set at 95% for both genders. Percent morphologically normal (%NORM) (Nomarski 1000X); percent total motility (TMOT), curvilinear velocity (VCL [um/sec]) (IVOS II); percent viability (VIAB; NucleoCounter) were evaluated at times, T1-after cooling, T2- after Equipure, T3- 0 to 6hs after sorting. Semen was cooled to 8°C and TMOT, VCL and VIAB were assessed at 18-24h (T4) and 48h (T5) post-sorting. Treatment means at each treatment/time were analyzed by a repeated measures mixed-model ANOVA. Sort quality measures (% DEAD) Peak Valley Ratio [PVR] and % percent of sperm correctly oriented [ORT]) were correlatedwith %NORM. Although there was a wide variation between stallions, 29/32 (91%) stallions were successfully sorted. The %NORM (mean±sd) was 59±11, 74±9 and 77±9% for T1, T2 and T3 respectively (p<0.05). For TMOT, T1 (67%) was less than T2 (74%; P<0.05) and similar to T3 (71%; P>0.05); for VIA, T1 (74%) was less than T2 and T3 (79 and 80%; P<0.05); for VCL, T1 (157µ/s) was less than T2 (171 µ/s; P<0.05) and similar to T3 (166 µ/s; P>0.05). For sperm longevity (TMOT, VIA, and VCL) T4 (49, 73, 147) and T5 (27, 63, 117) decreased, compared to T3. The initial quality of the semen and the number of morphological abnormalities had a high impact on the sorting and quality parameters ORT and PVR were positively correlated while DEAD was negatively correlated with %Norm cells in T1, T2 and T3. In this study, the SS process (cooling, colloid centrifugation and SS) occurred over an ∼ 24-hour period and the quality of the SS sperm was improved and maintained over that period which is similar to those normally utilized for conventional cool-shipped semen programs. Current sex-sorting technology has improved significantly, resulting in a high number of stallions sorting successfully, allowing broad application of gender selection technology to the equine breeding industry.

HCG versus histrelin acetate: ovulation inducing agents in mares (Equus caballus) in a commercial breeding program in Uruguay

Hormonal control of ovulation timing is an effective tool to optimize reproductive management and reduce costs in equine breeding programs. Few studies have evaluated the efficacy of GnRH analog histrelin as an ovulation inducer agent. This study aimed to compare the efficacy of human chorionic gonadotrophin (hCG) and histrelin as ovulation inducers in young, adult, and old mares during spring and summer. Reproductive records of 321 estrous cycles of 125 mares, mean age 9.8±0.37 years, range 1.5–24 years, between 360-600 kg body weight, and body condition ≥3 (1/5), were evaluated during spring (151 cycles) and summer (170 cycles). Mares were kept in the field and fed alfalfa hay, balanced concentrate feed and ad-libitum water. The ovaries, uterus, and cervix underwent transrectal examination and ultrasonography daily. When only one dominant follicle of 35 mm and endometrial edema compatible with estrus were reached, ovulation was induced. Each cycle was monitored every two to six hours until ovulation, 24h after induction. Cycles were randomly allotted in two groups; G1 (n=106 cycles): 1500 IU hCG (IV); G2 (n=215 cycles): 250 μg histrelin acetate (IM). A total of 119 cycles were from young (<8 years), 93 from adults (8-15 years), and 109 from old mares (>15 years). Cycles of mares ovulating within 24-48hafter treatment were considered to have responded to the inducer, and those ovulating before 24h and after 48h were excluded from the experiment. Inducer agents, season (spring or summer), and mare age were independent factors, and ovulation between 24-48h was considered the dependent factor. Data were analyzed through Fisher's exact test. The significance level was defined as P<0.05. From the 321 induced cycles, 254 (79.1%) ovulated within 24-48h, 20 (6.2%) before 24h, and 47 (14.6%) after 48h. There were no differences (P=0.2414) in the average response time between G1 (37.2h±3,5) and G2 (39.3h±4,5). The ovulatory response between G1 and G2 did not differ in spring (84.1% versus 90.7%, P=0.2627) and summer (79.2% versus 85.4%, P=0.3534). Independent of the inducer, the season did not influence the ovulation response in the different mare ages (spring P=0.1489; summer P=0.2992). No differences (P=0.896) were found between drugs in young and adult mares. However, old mares had a higher percentage of ovulatory response in spring and summer to histrelin (n=76) than to hCG (n=24) (89.5% versus 45.8%, P<0.0001). In conclusion, histrelin is more effective and accurate in inducing ovulation in old mares (>15 years) than hCG throughout the breeding season.

An alternative method to circumvent poor semen cooling and fertility of stallions

Cooled-shipped semen is routinely used in the equine breeding industry. Although most stallions present satisfactory semen quality and fertility after cooling and transportation, a population of stallions offer a suboptimal sperm cooling tolerance even when strategies to enhance stallion semen cooling are applied. Therefore, it is critical to developing alternative methods to improve the post-cooling semen quality from this population of stallions. This study assessed sperm parameters and fertility of cooled-stored stallion semen processed and resuspended in three extenders. In experiment 1, semen was collected from 21 stallions classified as having good (“Good coolers,” n=8) or poor (“Bad coolers,” n=13) sperm characteristics during cooled-storage after semen dilution in milk-based extender (SM, BotuSemen®). Immediately after semen collection, semen was extended at 50 million/mL in SM extender and stored at 5°C for 24h. Thereafter, cooled-stored semen was processed by SpermFilter® or conventional centrifugation (600 × g/10 min), and the resulting pellets were resuspended in SM, SM containing pentoxifylline (SM-P, BotuSemen Turbo®), or egg yolk-based extender containing cryoprotectants (EY, BotuCrio®). Unprocessed cooled semen served as control. Sperm motility parameters, plasma membrane integrity (PMI), and mitochondrial membrane potential (HMMP) were assessed in cooled-semen pre- and post-processing. In experiment 2, 66 cooled semen doses from stallions (n=9) classified as Bad coolers were randomly used to inseminate18 mares in 66 cycles (Unprocessed, n=22; SpermFilter®/SM-P, n=16; or SpermFilter®/EY, n=28 cycles). Data were analyzed with a mixed model, Tukey's, and logistic regression. Overall, resuspending centrifuged (Total motility [TM], 73±14%; progressive motility [PM], 36±13%) or filtered (TM, 75±15%; PM, 37±13%) semen in EY enhanced (P<0.05) sperm motility compared to unprocessed semen (TM, 50±33%; PM, 23±18%) and the other groups. However, when semen from only Bad Coolers was analyzed, motility of semen processed by SpermFilter® and resuspended in SM-P was enhanced (TM, 52±19%; PM, 22±14%) compared to unprocessed semen (TM, 30±26%; PM, 10±10%) and similar (P>0.05) to semen resuspended in EY (TM, 69±16%; PM, 34±14%). Pellet resuspension with EY (52±25%) and SM-P (49±25%) improved the HMMP of Bad cooler stallions (P<0.05) compared to unprocessed semen. Semen processed by SpermFilter® had superior PMI (72±8%) than centrifuged semen (64±11%; P<0.05) overall. In experiment 2, pregnancy rate of mares inseminated with SpermFilter®/SM-P (50%) or SpermFilter®/-EY (68%) was greater than with unprocessed semen (14%) (P<0.05). In conclusion, semen parameters and fertility of stallions with poor semen cooling can be enhanced by SpermFilter® and resuspension of the pellets with EY or SM-P. Acknowledgments: The authors thank FAPESP (grant #2017/13883-9) for the financial support.

In vitro reduction of Escherichia coli biofilm mass by platelet-rich plasma lysate (PRP-L)

Placentitis-induced, pre-term fetal loss has caused hardships and financial losses to the equine breeding industry for decades. While traditional multi-modal treatments including a combination of gentamicin, penicillin, altrenogest, firocoxib, and pentoxifylline can sometimes prolong gestation and neonatal survival, treatment failures still occur and antibiotic therapy can be quite lengthy. Understanding potential biofilms in these reproductive infections uncovers a pitfall to the success of traditional treatment options: antimicrobials are ineffective at penetrating and killing biofilms in vitro and in vivo. Platelet-rich plasma lysate (PRP-L) is increasingly accepted as an adjunctive treatment option for biofilm associated infections across species. The aim of this study was to evaluate the reduction in biofilm formation of 10 isolates of Escherichia coli collected from the equine uterus. Isolates were stored in glycerol at -80°C; each isolate including a positive (ATCC 25922) and negative (ATCC BAA 2469) strain, was cultured for 18-24 h at 33-37°Cin ambient air until bacterial colonies were visible. A suspension of each isolate, adjusted to a 0.5 McFarland value, was used to inoculate a tissue culture treated, round-bottom 96-well microtiter plate incubated under microaerophilic orbital shaking conditions for 18-24 h at 33-37°C. Following incubation, with or without PRP-L, the supernatant was removed, wells were stained with 0.03% crystal violet for 15 minutes at room temperature with orbital shaking, rinsed to remove excess stain, solubilized with ethanol, and absorbance optical density (OD570) read with a plate reader. The range in reduction of biofilm mass with PRP-L treatment was 48 – 84%, with an average of 60%. In conclusion, the results suggest that comparison of a traditional multi-modal therapy for placentitis with a multi-modal therapy augmented with PRP-L has the potential to finally improve placentitis outcomes and reduce the length of antibiotic treatment, but further in vivo evaluation of this approach is required.