T. Chenier

Equine Disorders of Sexual Development in 17 Mares Including XX, SRY-Negative, XY, SRY-Negative and XY, SRY-Positive Genotypes

We described the clinical, cytogenetic and molecular findings of 17 clinical equine cases presented for abnormal sexual development and infertility. Six horses with an enlarged clitoris had an XX, SRY-negative genotype, which displayed male-like behavior (adult individuals). Bilateral ovotestes were noted in 2 of those cases, while another case showed increased levels of circulating testosterone. Six horses with a female phenotype, including normal external genitalia, had an XY, SRY-negative genotype. These individuals had small gonads and an underdeveloped internal reproductive tract. Four horses with normal appearing external genitalia had an XY, SRY-positive genotype, 3 of them had hypoplastic testes and male-like behavior. In addition, one young filly with enlarged clitoris and hypoplastic testes had the same genotype but did not show male-like behavior due to her age. Three of these horses were related with 2 being siblings. These findings demonstrate the diversity of disorders of sexual development seen in the horse. Furthermore, they emphasize the need for further research to identify genes involved in abnormal sex determination and differentiation in the horse.

Assessment of stallion spermatozoa viability by flow cytometry and light microscope analysis

Viability of spermatozoa can be assessed by numerous methods, but many are slow and poorly repeatable, and subjectively assess only 100 to 200 spermatozoa per ejaculate. We collected two ejaculates from each of 4 stallions, and extended them to 50x10(6) sperm/mL in a nonfat dried milk solids glucose extender (EZ Mixin). Half the ejaculate was freeze-killed by immersing in liquid nitrogen for 10 min. Aliquots using appropriate volumes of live and freeze-killed spermatozoa provided the following ratios of live:dead spermatozoa: 100:0, 75:25, 50:50, 25:75, 0:100. We determined the viability of each aliquot by 1) motility; 2) eosin-nigrosin staining; and 3) dual fluorescent staining. For the latter, aliquots incubated with SYBR-14 and propidium iodide had live and dead spermatozoa quantitated by fluorescent microscope (2 x 100 sperm/sample) and flow cytometry (10,000 sperm/sample). We found a linear relationship between the ratio of live:dead spermatozoa and the percentage of spermatozoa counted as live (P<0.0001). For fresh spermatozoa, correlation coefficients of the known live:dead ratio were high for all methods (eosin-nigrosin, r>0.75; fluorescent microscope, r>0.76; flow cytometry, r>0.75; motility, r>0.76). To determine viability of cryopreserved equine spermatozoa, we froze 17 fresh ejaculates from 6 stallions in a glycine extender. Each sample was thawed, extended 1:1 with EZ Mixin and evaluated as above. Cryopreserved spermatozoa assessed by flow cytometry tended to be less well correlated (r<0.68) with the other methods, and estimates were significantly higher with eosin-nigrosin staining (P<0.001). This study shows that different methods may equally estimate viability of fresh equine spermatozoa. However, evaluation by flow cytometry appears to be less precise with cryopreserved spermatozoa.

GTG Mutation in the Start Codon of the Androgen Receptor Gene in a Family of Horses with 64,XY Disorder of Sex Development

Genetic sex in mammals is determined by the sex chromosomal composition of the zygote. The X and Y chromosomes are responsible for numerous factors that must work in close concert for the proper development of a healthy sexual phenotype. The role of androgens in case of XY chromosomal constitution is crucial for normal male sex differentiation. The intracellular androgenic action is mediated by the androgen receptor (AR), and its impaired function leads to a myriad of syndromes with severe clinical consequences, most notably androgen insensitivity syndrome and prostate cancer. In this paper, we investigated the possibility that an alteration of the equine AR gene explains a recently described familial XY, SRY + disorder of sex development. We uncovered a transition in the first nucleotide of the AR start codon (c.1A>G). To our knowledge, this represents the first causative AR mutation described in domestic animals. It is also a rarely observed mutation in eukaryotes and is unique among the >750 entries of the human androgen receptor mutation database. In addition, we found another quiet missense mutation in exon 1 (c.322C>T). Transcription of AR was confirmed by RT-PCR amplification of several exons. Translation of the full-length AR protein from the initiating GTG start codon was confirmed by Western blot using N- and C-terminal-specific antibodies. Two smaller peptides (25 and 14 amino acids long) were identified from the middle of exon 1 and across exons 5 and 6 by mass spectrometry. Based upon our experimental data and the supporting literature, it appears that the AR is expressed as a full-length protein and in a functional form, and the observed phenotype is the result of reduced AR protein expression levels.

Associations between feed efficiency, sexual maturity and fertility-related measures in young beef bulls

The beef industry has emphasized the improvement of feed utilization, as measured by modeling feed intake through performance traits to calculate residual feed intake (RFI). Evidence supports an inverse relationship between feed efficiency and reproductive function. The objective of this study was to determine the relationship of reproductive assessments and RFI unadjusted (RFI(Koch)) or adjusted for body composition (RFI(us)) and the relationship among fertility-related parameters. In total, 34 crossbred bulls were housed together for 112 days of performance evaluation, followed by assessment of scrotum IR imaging, scrotal circumference, testes ultrasonography and semen quality parameters at 377±33.4 days of age. Bulls were slaughtered at 389±34.0 days of age, and analyses of carcass composition, biometrics and histomorphometry of the testis and epididymis were conducted. Bulls were grouped into two subpopulations based on divergence of RFI, and within each RFI model either by including 50% of the population (Halves, high and low RFI, n=17) or 20.6% extremes of the population (Tails, high and low RFI, n=7). The means of productive performance and fertility-related measures were compared through these categories. Pearsons correlation was calculated among fertility-related measures. In the Halves subpopulation of the RFI(us), sperm of low-RFI bulls had decreased progressive motility (47.30% v. 59.90%) and higher abundance of tail abnormalities (4.30% v. 1.80%) than that of high-RFI bulls. In the Tails subpopulation of the RFI(Koch), low RFI displayed less variation in the scrotum surface temperature (0.62°C v. 1.16°C), decreased testis echogenicity (175.50 v 198.00 pixels) and larger (60.90 v. 56.80 mm(2)) but less-developed seminiferous tubules than high-RFI bulls. The evaluation of fertility-related parameters indicated that a higher percentage of immature seminiferous tubules was correlated with occurrence of sperm with distal droplets (r=0.59), a larger temperature variation at the top of the scrotum was correlated with improved sperm progressive motility (r=0.38), a lower occurrence of sperm loose head abnormalities was correlated with larger temperature variation at the lower part of the scrotum (r=-0.43), and a lower minimum testis echogenicity (r=-0.59) and smaller scrotal circumference (r=0.72) were correlated with age. The adjustment for body composition (RFI determination) enabled distinct biological inferences about reproduction and feed efficiency when compared with the non-adjusted model. However, both RFI models and the correlation analysis supported the hypothesis that feed-efficient bulls have features of delayed sexual maturity. Overall, the assessment of fertility-related measurements is important to avoid the improvement of feed efficiency at the expense of reproductive function in young bulls.

Treatment of Hydropsical Conditions Using Transcervical Gradual Fetal Fluid Drainage in Mares With or Without Concurrent Abdominal Wall Disease

&NA; Hydropsical conditions are exceedingly rare in the horse. However, when they occur, they are true emergencies due to the severe enlargement of the pregnant uterus, which can result in clinical signs, such as an enlarged round abdomen, dyspnea, reluctance to walk, and colic, and may lead to the development of abdominal wall disease. The pathogenesis of hydropsical conditions is not fully elucidated, but they have been associated with placentitis and fetal abnormalities. This report describes six cases of hydropsical conditions in mares with or without concurrent abdominal wall disease. Five out of six cases were hydrallantois, and of these five, two mares had abdominal wall disease; the remaining one out of six cases was hydramnios. All mares were treated by termination of the pregnancy through gradual fluid drainage transcervically over a number of hours, and their fetuses were delivered vaginally. All fetuses were euthanized immediately after vaginal delivery. Of the six mares, two had signs of placentitis, two were confirmed seropositive for leptospirosis, and two were euthanized (one because of a vaginal tear that communicated through the peritoneum and one mare that developed abdominal wall rupture and laminitis). The remaining 4 mares were available for follow‐up; three mares were not rebred, and one mare became an embryo donor, with a successful embryo recovery. We reported the prevalence of leptospira involvement in two out of six cases of hydrallantois and also described the clinical outcome of the mares after treatment with slow fetal fluid drainage.

Relationships of nutritional plane and feed efficiency with sexual development and fertility related measures in young beef bulls

Sexual development in beef bulls appears to be influenced by nutritional plane and feed efficiency. Yearling bulls fed high (GRAIN = 49) and moderate (ROUGHAGE = 109) nutritional planes, were submitted to a performance test where scrotal circumference (SC), scrotum and testis ultrasonograms and hormones were monitored throughout. Scrotal thermographs, blood cell counts and semen were evaluated at the end of the test. Residual feed intake (RFI) was the measure of feed efficiency, and bulls within each population were characterized as efficient and inefficient. During the test period, the GRAIN group had greater triiodothyronine (T3), leptin and scrotal skin thickness (SST) while having greater T3, leptin, % motile sperm, % normal sperm, scrotal temperature and values for red blood cell variables at the end of the test when compared to the ROUGHAGE group. During the test, the efficient GRAIN group had lesser testis pixel intensity while at the end of the performance test the bulls of this group had greater % normal sperm and lower testis pixel intensity compared to bulls in inefficient GRAIN group. In comparison, the efficient ROUGHAGE group had less T3, SC and SST while at the end of the test these bulls had less T3, leptin, SC, SST and scrotal temperature compared to bulls in the inefficient ROUGHAGE group. Complete blood cell variables, semen quality, scrotal biometry and thermography explained most of the variation in RFI. Results of the present study support the hypothesis that there is an antagonistic relationship between feed efficiency and sexual development in young bulls, which seems partially offset when there is a greater plane of nutrition. Thus, feed efficiency should be factored in the reproductive evaluation of sires.

Seminal plasma protein concentrations vary with feed efficiency and fertility-related measures in young beef bulls

Fertility-associated proteins (FAP) found in seminal plasma indicate sexual maturity, which appears to be influenced by feed efficiency in cattle. This study characterized FAP via proteomics and verified associations of these proteins with feed efficiency, body composition and fertility-related measures in yearling beef bulls. Assessments including testicular ultrasonography, infrared thermography, seminal quality, seminal plasma proteomics, carcass composition, and reproductive organ biometry were obtained. From a population of 31 bulls, the seven most and least feed efficient (efficient, inefficient) bulls were used for categorical comparisons. Correlations between FAP, productive performance and fertility-related measures were determined. These traits were also correlated with orthogonal factors summarized from the FAP. Efficient bulls had increased epididymal sperm-binding protein-1 and decreased concentration of protein-C inhibitor compared to inefficient bulls. Correlations between FAP with age, body size, body composition, reproductive organ biometry, scrotal temperature, and seminiferous tubule maturity are reported. Acrosin and cathepsin D increased with development of the testes and osteopontin increased with greater numbers of mature seminiferous tubules. Phosphoglycerate kinase-2 was higher in animals with a higher scrotum temperature and a higher prevalence of sperm morphology defects. The principal factor indicated that FAP variability concentrations were positively correlated with age, reproductive organ biometry, body size and composition. Our results indicate that FAP changes with body size and sexual development, and demonstrates differences in the proteomics of bulls with diverging feed efficiency. This is related to the delay in the sexual maturity of efficient young bulls.