The goal of this study was to compare the efficacy of coated iron-core nanoparticles and single-layer centrifugation for separation of dead from live stallion spermatozoa. Our hypothesis was that nanoparticles would bind to dead sperm and allow for separation from live sperm using a magnet, resulting in a population of spermatozoa with a high percentage of total and progressive motility. Treatment Group 1 was an untreated control. Treatment Group 2 (nanoparticles, NP) utilized sperm incubated with nanoparticles followed by application of a magnet to remove dead sperm adhered to the coated nanoparticles. Treatment Group 3 (single-layer centrifugation, SLC) layered sperm above EquiPure™ followed by centrifugation. Semen samples were subsequently evaluated for sperm motility parameters, plasma membrane integrity, acrosome status, and morphology. The SLC technique yielded higher (p < 0.05) progressive motility (76 ± 9.2%) than the NP separation technique (59 ± 12.2%) or the untreated control (47.3 ± 5.1%). However, the total number of sperm recovered was higher (p < 0.05) in the NP technique (526.2 ± 96.6 × 106) than the SLC procedure (211.7 ± 70 × 106), yielding a higher total number of progressively motile sperm (317.6 ± 109 × 106) recovered using the NP technique than the SLC technique (157.8 ± 43.6 × 106). The percentage of live, acrosome intact sperm recovered was higher for SLC than NP. In summary, the SLC technique yielded a higher percentage of sperm motility, intact plasma membranes, and acrosome integrity, but yielded lower total sperm than with the nanoparticle separation technique.

β-Nerve growth factor (β-NGF) is a protein produced in the reproductive tract of camelids (camels, llamas, and alpacas) that has been identified as the ovulation inducing factor in seminal plasma. β-NGF from seminal plasma deposited into the reproductive tract of the female camelid acts systemically to stimulate the secretion of luteinizing hormone (LH) from the anterior pituitary, which in turn induces follicle maturation and ovulation. The objectives of the present study were to determine if β-NGF is present in the reproductive tract of the stallion and identify the specific site(s) of production. The hypotheses were that β-NGF would be present in the stallion reproductive tract and would primarily be localized in Sertoli cells of the testes and the prostate gland. Immunohistochemistry on paraffin-embedded paraformaldehyde-fixed tissues was performed using a rabbit polyclonal anti-β-NGF antibody on a total of six male equine reproductive tracts, including a one-day old colt, a one-year-old colt, and four adult stallion tracts. Strong immunostaining was observed in the efferent ducts of the testes and the epithelial cells of the prostate, seminal vesicles, bulbourethral glands, and ampullae. Weaker β-NGF staining was noted in Leydig cells, Sertoli cells, and spermatogonia within the testes and in epithelial cells of the epididymis. In conclusion, immunohistochemistry revealed that β-NGF is present in the stallion reproductive tract, and the protein is primarily present in the efferent ducts of the testes and in all accessory sex glands.

BackgroundGenetic testing is required for the registration of foals of most equine breeds. ObjectivesTo describe two clinical cases of marked delayed embryonic development or delayed fertilisation in pregnancies generated by embryo transfer. Study designCase report. MethodsDonor mares were inseminated with semen from one stallion during one oestrous cycle and semen from a different stallion on the subsequent oestrous cycle. Embryo(s) were collected 8 days after ovulation during the second oestrous cycle and transferred into synchronised recipient mares. Genetic testing was performed to determine parentage of the two foals. ResultsFor both foals, DNA parentage testing excluded the second stallion as the genetic sire and confirmed that the first stallion, whose semen was inseminated on the previous oestrous cycle, was the actual genetic sire. Main limitationsRare event in horses; two clinical cases are described. ConclusionsIt is hypothesised that either marked delayed embryonic development or extended sperm survival occurred in the donor mares. Without genetic testing, parentage assignment based solely on breeding records would have been incorrect.

Inducing ovulation of multiple follicles has been an elusive goal in equine reproduction. Administration of porcine FSH, ovine FSH or GnRH agonists only yield a marginal increase in ovulation rate in mares. Early studies utilizing single chain recombinant equine FSH (reFSH) reported stimulation of follicular development in anestrous mares and increased ovulation rates in cycling mares. Unfortunately, the original reFSH product from AspenBio Pharma, Inc. (Castle Rock, CO USA) is no longer in production. The goal of the current study was to evaluate the efficacy of a new single chain recombinant equine FSH (reFSH-FP; FertilPlus Partners, LLC; Conroe, TX USA) in stimulation of follicular development in mares in two pilot studies as compared to the original reFSH (reFSH-AB; AspenBio Pharma). Study 1. Nine seasonally anestrous mares (follicle diameter 19 ± 3.7 mm) were randomly assigned to one of 3 treatment groups (n=3/group): saline placebo, reFSH-AB (0.65 mg, IM, q12), reFSH-FP (0.65 mg, IM, q 12h) for a maximum of 7 days. A dose of hCG (2,500 units, IV) was administered after a 36-hour coast period once one or more follicles attained a diameter of ≥ 35 mm. The ovulation rates for the three treatment groups were 0, 10.3 ± 3.5, and 6.0 ± 2.0 ovulations for the saline placebo, reFSH-AB and reFSH-FP, respectively. Study 2. Twelve normally cycling mares were randomly assigned to one ofthe same treatment protocols as described for Study 1 above (n=4 per group). All mares were in mid-diestrus at the onset of treatment and a dose of cloprostenol (250 µg, IM) was administered on the second day of treatment. Mares were treated for a maximum of 7 days. HCG (2,500 units, IV) was administered after a coast period of 36 hours when one or more follicles attained a diameter of ≥ 35 mm. Ovulation rates were 1.3 ± 0.5, 5.5 ± 5.3, and 5.3 ± 1.0 ovulations for the saline placebo, reFSH-AB and reFSH-FP, respectively. All mares ovulated in the placebo and reFSH-FP groups after administration of hCG; one mare in the reFSH-AB group developed 12 follicles > 35 mm in diameter, but failed to ovulate after administration of hCG. Collectively, these studies confirm that the new recombinant equine FSH product (reFSH-FB; FertilPlus) has biological activity in the mare similar to that of the original reFSH product (reFSH-AB; AspenBio Pharma) and is capable of stimulating development of multiple large follicles in deep anestrus and cycling mares. However, significant hurdles remain before a recombinant equine FSH product could be available for commercial production, distribution and clinical use. Additional research will be needed to evaluate safety, dosage, frequency of administration, timing of administration of an ovulation induction agent, and fertility following reFSH treatment.

Disorders of sexual development (DSD) are associated with atypical chromosomal, gonadal, or phenotypic sex. It is likely that the number of cases of DSD are underestimated in the equine population. Monorchidism in the horse is very rare. This case report describes the clinical assessment of a phenotypic mare with stallion-like behavior which led to the diagnosis of a DSD. A 4-year-old Quarter Horse mare presented in good body condition, with normal external genitalia for a mare, and normal mammary glands with two bilaterally symmetric teats. No uterus, cervix, or gonads were detected on transrectal palpation. Transrectal ultrasonography revealed a single gonad in the right dorsal abdomen with the morphologic appearance of a testicle. Presurgical hormonal evaluation revealed elevated serum testosterone and anti-Müllerian hormone (AMH) concentrations. The right gonad was successfully removed via standing exploratory laparoscopy and submitted for histopathology. No gonad was identified on the left side during laparoscopy. Histopathologic examination confirmed that the excised gonad was a testicle. Cytogenetic and molecular analysis revealed a 64,XY, SRY-positive chromosomal constitution. Hormonal evaluation 5 weeks after surgery revealed low serum testosterone and AMH levels. A diagnosis of monorchidism was based on ultrasound examination, laparoscopic exploration of the abdomen, removal of a single gonad, and a subsequent decrease in serum testosterone and AMH concentrations to basal levels. In summary, a combination of clinical signs, endocrine evaluation, chromosomal and molecular analysis, and histopathology can be used in the diagnosis of DSD conditions.

Congential amastia, a medical condition in which mammary tissue fails to develop, was detected in a 3-year-old Quarter Horse mare. The dam of the mare was also afflicted with amastia, suggesting that the condition was due to an inherited genetic mutation as noted in other species. In addition, on presentation the mare had a purulent vaginal discharge secondary to a pyometra.

An embryo collection procedure is usually performed 7-8 days post-ovulation in mares. Embryos are subsequently transferred into a synchronized recipient mare that is expected to carry the pregnancy to term. Submission of a blood or hair root sample to an approved genetics laboratory for DNA parentage verification is a prerequisite for foal registration for most horse breed organizations. The goal of this report is to document two clinical cases in which the DNA parentage test of the foals produced by embryo transfer excluded the stallions whose semen was used on the estrous cycle yielding the embryo and identified as the genetic sire the stallion whose semen was utilized on the previous estrous cycle. Clinical Case # 1. A 21-year-old Arabian mare was inseminated with frozen-thawed semen from a deceased stallion (Stallion A) immediately after detection of a single ovulation. No embryo was recovered following uterine lavage 8 days later. Cooled-transported semen from a different stallion (Stallion B) was used on the subsequent cycle. An exceptionally large (2,466 µm in diameter) expanded blastocyst stage embryo was recovered 8 days post-ovulation and transferred into a recipient mare. The recipient mare carried the pregnancy to term. Genetic testing of the foal excluded Stallion B as the sire and confirmed Stallion A as the genetic sire of the foal. Clinical Case # 2. A 11-year-old Quarter Horse marewas inseminated with cooled-transported semen from Stallion A and subsequently ovulated two follicles. Uterine lavage 6.5 days later yielded one embryo. Cooled-transported semen from a different stallion (Stallion B), housed at a different reproductive center, was used on the second cycle. The mare ovulated one follicle and uterine lavage 8 days post-ovulation yielded two embryos, one of which was substantially larger than expected. The recipient mare receiving the larger embryo carried the pregnancy to term. DNA parentage testing excluded Stallion B as the genetic sire and confirmed Stallion A as the genetic sire. The intervals from the dates of the original insemination with the genetic sire to the first cycle ovulation, second cycle ovulation and recovery of the embryos in question were 0 days (post-ovulation insemination), 18 days, and 26 days for the Arabian mare and 2 days, 18 days and 26 days for the Quarter Horse mare. The potential explanations for how an equine embryo could be recovered following an insemination 26 days previously include marked delayed fertilization or marked delayed embryonic development. Equine DNA parentage testing relies on the principle of exclusion, with the inheritance of a series of short tandem repeat (STR or microsatellite) markers evaluated in the foal, sire, and embryo donor mare. Parentage assignment based solely on breeding records would have been incorrect.

The energy source for motility is produced in the mitochondria of the middle piece of a spermatozoon. Rhodamine 123 (R123) is the most widely used mitochondrial probe. The lipophilic probe JC-1 has been reported as distinguishing sperm cells with high or low mitochondrial membrane potential. JC-1 is excited by visible light (488 nm) and accumulates in the mitochondria of the sperm. This chapter discusses technique for the assessment of sperm mitochondrial function using JC-1 and R123. With the use of JC-1, two distinct sperm populations can be observed. A higher percentage of functioning mitochondria in the sample provides information that the sperm are able to produce the energy needed for motility. Changes in the procedures used to cryopreserve the sperm cells may help to increase the amount of cells with high mitochondrial membrane potential after thawing.

Stallion spermatozoa for intracytoplasmic sperm injection (ICSI) can be acquired from fresh, cooled-transported, frozen, refrozen, and even lyophilized semen. This chapter discusses laboratory techniques for processing semen for ICSI. A majority of clinical ICSI procedures are performed using frozen-thawed semen from a stallion selected by the owner of the oocyte donor mare. The most common techniques for processing frozen stallion semen for ICSI are swim-up, washing, single layer centrifugation, density gradient centrifugation, microfluidic sorting, or a combination of the procedures. In addition, some ICSI laboratories tailor sperm processing procedures for individual stallions. After the final wash, sperm pellets are resuspended in synthetic oviductal fluid–in vitro fertilization medium and subsequently diluted in polyvinylpyrrolidone 12% in phosphate buffered saline before ICSI.

A stallion is considered to be a cryptorchid if one or both testes are not fully descended into the scrotum. Cryptorchid testes may be located anywhere from the caudal pole of the kidneys to adjacent to the inguinal rings. An accurate diagnosis of cryptorchidism is not difficult if the horse has had one owner and if adequate medical records have been maintained. Diagnostic evaluation of a male horse that may have one or two retained testes should begin with collection of a complete medical history, including age, breed, previous ownership, previous veterinary examinations, surgical procedures, and diagnostic tests. The absence of a history of castration in a male horse with either one or no scrotal testes is immediately indicative of unilateral or bilateral cryptorchidism, respectively.

Gonadotropin-releasing hormone (GnRH) produced in the hypothalamus stimulates the secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the anterior pituitary. Pulses of GnRH induce pulsatile secretion of LH and FSH which, in turn, regulate testicular function. Administration of GnRH to a stallion will stimulate the synthesis and secretion of LH and FSH and may be used as a test of pituitary function. Test strategies include the administration of a physiological dose of GnRH to measure pituitary function and the administration of a pharmacological dose of GnRH to measure pituitary content of LH and FSH. In either situation, it is recommended that a normal fertile stallion be tested along with a subfertile stallion to evaluate and compare pituitary responses. The low dose, single pulse GnRH stimulation test is used to assess pituitary and potentially testicular responsiveness.

The natural or physiological breeding season of mares extends from April to October in the northern hemisphere. A majority of mares enter a state of ovarian inactivity, or seasonal anestrus, during the winter months. This chapter gives step-by-step instructions with interpretative information, as well as useful equipment lists for management of seasonal anestrus. Mares housed under a stimulatory artificial photoperiod will ovulate earlier in the year and potentially cycle and ovulate more often during the breeding season than mares maintained under ambient light conditions. The physiological mechanism by which artificial photoperiod works is through interruption of the normal pattern of melatonin secretion from the pineal gland. Melatonin is secreted during the hours of darkness. Extending the duration of light exposure will reduce the duration of melatonin secretion, which will subsequently stimulate hypothalamic production of gonadotropin-releasing hormone.

Infection of the seminal vesicles of stallions is uncommon, but when present can be challenging to treat. Systemic antibiotic therapy is not often successful as most antibiotics do not diffuse across the internal mucosal border of the seminal vesicles. Another option for treatment is irrigation of the affected seminal vesicle(s) using a cannula passed through a videoendoscope, followed by infusion of antibiotics directly into the affected vesicle. The chapter discusses the technique for endoscopic-guided cannulation of the seminal vesicles. An additional management procedure for stallions with seminal vesiculitis is collection of semen and subsequent dilution with an extender containing an appropriate antibiotic based on an antimicrobial susceptibility test.

Ultrasound examination of the reproductive tract is the most reliable test for pregnancy diagnosis. A thorough, systematic approach to ultrasonographic pregnancy diagnosis is imperative in order to correctly diagnose pregnancy status and to detect twins. This chapter discusses the processes involved in the ultrasound examination of the reproductive tract. The frequency and schedule of initial and subsequent examinations are often dictated by factors such as availability of the mare or clinician, potential for twins, results of previous pregnancy examinations, a history of pregnancy loss in a particular mare, and economics. A routine follow-up examination by ultrasonography is generally recommended between 25 and 35 days post-ovulation to confirm that the pregnancy is ongoing and that a heart beat is present.

Chapter 104 Evaluation of Colostrum Quality Brix Refractometry Patrick M. McCue, Patrick M. McCue Equine Reproduction Laboratory, Colorado State University, USASearch for more papers by this author Patrick M. McCue, Patrick M. McCue Equine Reproduction Laboratory, Colorado State University, USASearch for more papers by this author Book Editor(s):John Dascanio VMD, John Dascanio VMD Diplomate ACT and ABVP (Equine) Senior Associate Dean for Academic and Student Affairs Professor of Theriogenology School of Veterinary Medicine, Texas Tech University, Amarillo, Texas, United StatesSearch for more papers by this authorPatrick McCue DVM, PhD, Patrick McCue DVM, PhD Diplomate ACT Iron Rose Ranch Professor of Equine Reproduction Colorado State University, Equine Reproduction Laboratory, Fort Collins, Colorado, United StatesSearch for more papers by this author First published: 19 February 2021 https://doi.org/10.1002/9781119556015.ch104 AboutPDFPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShareShare a linkShare onFacebookTwitterLinked InRedditWechat Summary The evaluation of mare colostrum immediately after foaling and prior to nursing can be helpful in predicting if adequate passive transfer or failure of passive transfer is likely to occur. The evaluation of colostrum is also valuable in determining the quality of colostrum to be harvested and stored in a colostrum bank. Quantitative measurement of IgG levels in colostrum can be obtained by a radial immunodiffusion (RID) assay at a diagnostic laboratory. Qualitative assessment of colostrum can be performed using a sugar (Brix) refractometer, which measures the concentration of dissolved solids in a solution. Brix refractometer evaluation of equine colostrum has been shown to be highly repeatable and highly correlated with IgG levels as measured by RID assay. Equine Reproductive Procedures, Second Edition RelatedInformation

Evaluation of the mare reproductive tract begins with a thorough and systematic manual palpation of the ovaries, uterus, and cervix per rectum. This may be followed by ultrasound evaluation of the reproductive tract and other procedures. This chapter gives step-by-step instructions with interpretative information, as well as useful equipment lists for palpation of the reproductive tract. Certain anatomic features such as softness of a pre-ovulatory follicle, sensitivity of the ovary in the peri-ovulatory period, tone in the uterus and cervix, and the presence of a parovarian cyst, are easier to discern on palpation than on ultrasound. The best way to manually examine the uterus is to advance the palpation arm cranially within the rectum beyond the uterus and then retract the arm while sweeping a cupped hand in a caudal–ventral direction. Evaluation of cervical morphology and detection of cervical relaxation failure is important when formulating a reproduction management plan for an older maiden mare.

Manual palpation should be followed by a systematic ultrasound evaluation of the entire reproductive tract. This chapter gives step-by-step instructions with interpretative information, as well as useful equipment lists for ultrasound evaluation. Ultrasound is used in broodmares to visualize structures in the reproductive tract that cannot be palpated or differentiated on palpation per rectum, such as detection of echogenic follicular fluid, endometrial cysts, and free fluid within the uterine lumen. In addition, ultrasonography is valuable in the early diagnosis of pregnancy, management of twins, and detection of potential ovarian or uterine pathology. Ultrasound also allows for critical evaluation of the events leading to ovulation, such as changes in follicular characteristics and grading of uterine edema. Ultrasonography should be performed after manual palpation of the reproductive tract.