Endometritis: Managing Persistent Post-Breeding Endometritis.

Endometritis was rated as the third most common medical problem encountered in adult horses in North America. It is the leading cause of subfertility in broodmares and is a major contributor to economic loss in the horse breeding industry, with pregnancy rates reported to be as low as 21% in mares with severe endometritis. Endometritis may be categorized as: endometrosis (chronic degenerative endometritis), acute, chronic, active, dormant, subclinical, clinical, and persistent post-breeding. These classifications are not mutually exclusive, and mares may change categories within breeding seasons or estrous cycles or may fit in multiple classifications. This chapter will focus on discussing etiology and management strategies for mares affected by persistent post-breeding endometritis. Overall, these mares are considered subfertile but acceptable pregnancy and foaling rates can be achieved with appropriate breeding management.

Improved sperm cryosurvival in diluents containing amides versus glycerol in the Przewalski's horse (Equus ferus przewalskii).

Two studies were conducted to understand sperm cryosensitivity in an endangered equid, the Przewalski's horse (Equus ferus przewalski), while testing the cryoprotectant ability of formamides. The first assessed the toxicity of permeating cryoprotectants (glycerol, methylformamide [MF] and dimethylformamide [DMF]) to Przewalski's horse spermatozoa during liquid storage at 4°C. The second examined the comparative influence of three diluents (with or without formamides) on cryosurvival of sperm from the Przewalski's versus domestic horse. When Przewalski's horse spermatozoa were incubated at 4°C in INRA 96 with differing concentrations of glycerol, MF or DMF or a combination of these amides, cells tolerated all but the highest concentration (10% v/v) of MF alone or in combination with DMF, both of which decreased (P<0.05) motility traits. There was no effect of cryoprotectants on sperm acrosomal integrity. In the cryosurvival study, average sperm motility and proportion of cells with intact acrosomes in fresh ejaculates were similar (P>0.05) between the Przewalski's (67%, 84%, respectively) and domestic (66%, 76%) horse donors. Sperm from both species were diluted in lactose-EDTA-glycerol (EQ), Botu-Crio (BOTU; a proprietary product containing glycerol and MF) or SM (INRA 96 plus 2% [v/v] egg yolk and 2.5% [v/v] MF and DMF) and then frozen over liquid nitrogen vapor. After thawing, the highest values recovered for total and progressive sperm motility, acrosomal integrity and mitochondrial membrane potential were 42.4%, 21.8%, 88.7% and 25.4CN (CN=mean JC-1 fluorescence intensity/cell on a channel number scale), respectively, in the Przewalski's and 49.3%, 24.6%, 88.9% and 25.8CN, respectively, in the domestic horse. Although sperm progressive motility and acrosome integrity did not differ (P>0.05) among treatments across species, mitochondrial membrane potential was higher (P<0.05) in both species using EQ compared to BOTU or SM media. Additionally, Przewalski's stallion sperm expressed higher (P<0.05) post-thaw total motility in BOTU and SM compared to EQ, whereas there were no differences among freezing diluents in the domestic horse. In summary, Przewalski's stallion sperm benefit from exposure to either MF or DMF as an alternative cryoprotectant to glycerol. Overt sperm quality appears similar between the Przewalski's and domestic horse, although the total motility of cells from the former appears more sensitive to certain freezing diluents. Nonetheless, post-thaw motility and acrosomal integrity values for Przewalski's horse spermatozoa mimic findings in the domestic horse in the presence of INRA 96 supplemented with 2% (v/v) egg yolk and a combined 2.5% concentration of MF and DMF.

Effects of activation and assisted reproduction techniques on the composition, structure, and properties of the sauger (Sander Canadensis) spermatozoa plasma membrane.

The sperm plasma membrane is a multifunctional organelle essential to fertilization. However, assisted reproduction techniques often negatively affect this structure, resulting in reduced fertility. These reductions have been attributed to plasma membrane damage in a wide array of species, including fish. Considerable research has been conducted on the fish sperm membrane, but few have examined the effect of cryopreservation and other assisted reproduction techniques (ARTs) on not only membrane composition, but also specific characteristics (e.g., fluidity) and organization (e.g., lipid rafts). Herein, we determined the effects of three ARTs (testicular harvest, strip spawning, and cryopreservation) on the sperm plasma membrane, using Sauger (Sander canadensis) sperm as a model. To this end, a combination of fluorescent dyes (e.g., merocyanine 540, filipin III, cholera toxin subunit ß), liquid chromatography - mass spectroscopy (LC-MS) analysis of membrane lipids, and membrane ultracentrifugation coupled with plate assays and immunofluorescence were used to describe and compare sperm fluidity, membrane composition, as well as lipid raft composition and distribution among sperm types. Stripped sperm became more fluid following motility activation (40% increase in highly fluid cells characterized by a 2 × increase in fluorescence) and contained lipid rafts restricted to the midpiece. Testicular harvest yielded sperm with characteristics similar to stripped sperm. By contrast, cryopreservation impacted every aspect of membrane physiology. Two cell populations, one highly fluid and the other rigid, resulted from the freeze-thaw process. Cryopreservation reduced lipid raft cholesterol content by 44% and flotilin-2 (a lipid raft marker) was partially displaced owing to a decrease in buoyancy. Unlike stripped and testicular sperm, LC-MS analysis revealed increases in oxidative damage markers, membrane destabilization, and apoptotic signaling in cryopreserved sperm. Ultrastructural analysis also revealed widespread physical damage to the membrane following freeze-thaw. Sperm motility, however, was unrelated to any measure of membrane physiology used in this study. Our results demonstrate that ARTs have the potential to substantially affect the sperm plasma membrane, but not always detrimentally. These results provide multiple potential biomarkers of sperm quality as well as insight into sources of sub-fertility resulting from use of ARTs.

Effects of components of semen extenders on the binding of stallion spermatozoa to bovine or equine zonae pellucidae.

The effects of semen extender components on the ability of stallion sperm to bind to the zona pellucida (ZP) and the suitability of using bovine ZP for a ZP-binding assay for stallion sperm were investigated in a series of experiments. In Experiment I, binding of stallion sperm to both bovine and equine ZP was significantly increased when a skim milk-based extender (EZM) was used. In Experiment II, a threefold increase in sperm binding to ZP was observed when sperm were diluted in EZM compared with diluents, which contained no milk (TALP, LAC, and EmCare). In Experiment III, centrifuging the sperm through Percoll did not increase sperm binding to the ZP but did remove any positive effect of EZM on sperm-ZP binding. In Experiment IV, exposure of either sperm or ZP to EZM before co-incubation did not increase sperm binding to ZP. In Experiment V, sperm diluted in TALP containing skim milk, EZM, or INRA96 bound more efficiently to the ZP than sperm diluted in TALP without milk proteins. In Experiment VI, sodium caseinate, native phosphocaseinate, and caseinoglycopeptide increased sperm binding to the ZP. In conclusion, diluents containing milk or milk proteins markedly enhanced the number of sperm bound to both equine and bovine ZP.

Effect of glucose concentration and cryopreservation on mitochondrial functions of bull spermatozoa and relationship with sire conception rate.

Mitochondrial function is essential for sperm viability, not only from a sperm metabolism perspective, but also for improvement of sperm storage in liquid and frozen states. Bull sperm have notable metabolic variability with energy production for motility and subsequently for fertilizing capacity resulting from both glycolysis and oxidative phosphorylation. The objective of this study was to determine mitochondrial function of sperm using high-throughput Seahorse Analyzer technology in fresh semen and subsequent to freezing-thawing when there was incubation in media commonly used for sperm storage (relatively large glucose concentration) and female tract (relatively small glucose concentration). Additionally, there were determinations whether there were differences in values for fertility variables by regressing sire conception rate on values for mitochondrial variables when there was evaluation of semen from bulls with varying fertility. Media with larger concentrations of glucose inhibited mitochondrial function in fresh sperm, as indicated by less maximal oxygen consumption, spare respiratory capacity and coupling efficiency when compared to sperm in the media containing less glucose. Furthermore, there was greater (P <  0.05) mitochondrial function in cryopreserved-thawed compared to fresh samples with there being no effect of incubation media. These results indicate that mitochondrial damage from cryopreservation cannot be simply overcome post-thawing with glucose supplementation of bull semen incubation media. The increase in mitochondrial function is likely due to "non-productive" oxygen consumption to maintain the mitochondrial proton gradient. Furthermore, there was a negative association of mitochondrial proton leakage with sire conception rate indicating this could be a potential biomarker of bull fertility.

Evaluation of Biofilm Production by Escherichia coli Isolated From Clinical Cases of Canine Pyometra.

Many Escherichia coli (E. coli) strains produce biofilm that confers antimicrobial resistance. However, studies of biofilm production by E. coli from canine pyometra are lacking. Objectives were to elucidate the role of biofilm production by E. coli in pyometra by: (1) assessing the ability of E. coli to produce biofilm in vitro, and (2) confirming biofilm in situ. Endometrial biopsies were obtained from bitches with pyometra and preserved for microscopic analysis (n = 25). An endometrial swab was submitted for aerobic culture. Samples with confirmed E. coli were evaluated further for biofilm production in vitro and in vivo. Seventy percent of cases (16/23) resulted in pure growth of 1 or 2 E. coli strains, totaling 20 isolates. Fifteen isolates (15/20, 75%) had higher optical densities then negative controls (P < .05). On histopathology, all tissues exhibited endometrial inflammation and mucus was located within endometrial glands and occasionally overlying epithelium on 14 slides (14/16, 88%). Bacteria was noted in 50% of slides (8/16). During FISH acellular debris within the uterine lumen consistent with biofilm was noted on 94% of samples (15/16) and E coli was positively identified on all samples (15/15). Areas suggestive of the presence of biofilm were observed on all samples on scanning electron microscopy; but, bacteria consistent with E. coli were only visualized in 9 samples (9/16, 56%). In conclusion, we demonstrated that relevant strains of E. coli produce biofilm in vitro and in vivo, which may be considered in the development of new pyometra treatments aimed at disrupting these E. coli biofilm.

Testicular collections as a technique to increase milt availability in sauger (sander canadensis).

This study was conducted to compare quality and quantity of sperm collected from sauger (S. canadensis) using two collection methods: stripping alone and testicular tissue collection combined with stripping. Sperm were collected from sauger broodstock (n = 20) during the breeding season. Fish were randomly assigned to two sperm collection groups: (1) stripping once or (2) stripping twice before testicular tissue collection for obtaining additional sperm. Sperm motility variables, morphology, total number produced, and fertilization (%) were compared using the two collection methods. Testicular sperm had greater total motility (70.1 ± 2.1% compared with 44.3 ± 5.7%) but there were fewer morphologically normal cells (76.4 ± 1.3% compared with 92.8 ± 1.0%) compared to sperm collected using the stripping procedure. Sperm collection regimen utilizing testicular collections and sperm extractions in combination with stripping resulted in a ∼ten fold increase in total number of motile and morphologically normal sperm (39.5 ± 4.1 × 10 9) compared with the currently utilized two sequential sperm stripping collection procedures alone (3.6 ± 4.1 × 10 9 sperm). In large-scale studies (150,000 eggs), fertilization, using sperm collected from testicular tissues (1.0 × 105 motile sperm/egg), was similar to sperm collected with only the stripping procedure (71.2 ± 5.5 %, 81.2 ± 5.5 %, P = 0.265). The results of this study indicate testicular collection combined with sperm extractions allows for collection of sperm of a quantity and quality to maximize fry production and reduce the problems with lack of broodstock availability for sperm collection.

Sintered electrospun polycaprolactone for controlled model drug delivery.

Electrospinning has been used widely for drug delivery applications due to its versatility and ease of modification of spun fiber properties. Net drug loading and release is typically limited by the inherent surface-area of the sample. In a relatively novel approach, sintering of electrospun fiber was used to create a capsule favoring long-term delivery. We showed that electrospun polycaprolactone (PCL) retained its initial morphology out to 1042 days of in vitro exposure, illustrating its potential for extended performance. Sintering decreased the electrospun pore size by 10- and 28-fold following 56 and 57 °C exposures, respectively. At 58 and 59 °C, the PCL capsules lost all apparent surface porosity, but entrapped pores were observed in the 58 °C cross-section. The use of Rhodamine B (RhB, 479.02 g mol-1), Rose Bengal (RB, 1017.64 g mol-1) and albumin-fluorescein isothiocyanate conjugate from bovine serum (BSA-FITC, ~66,000 g mol-1) as model compounds demonstrated that release (RhB > RB ≫ BSA-FITC) is controlled both by molecular weight and available porosity. Interestingly, the ranking of release following sintering was 57 > 56 > 59 > 58 °C; COMSOL simulations explored the effects of capsule wall thickness and porosity on release rate. It was hypothesized that model drug adsorption on the available fiber surface-area (57 versus 56 °C) and entrapped porosity (59 versus 58 °C) could have also attributed to the observed ranking of release rates. While the 56 and 57 °C exposures allowed the bulk of the release to occur in <1 day, the capsules sintered at 58 and 59 °C exhibited release that continued after 12 days of exposure.

Technical Note: The use of iSperm technology for on-farm measurement of equine sperm motility and concentration.

The iSperm is a newly released semen analysis tool from Aidmics Biotechnology Co. LTD, which allows an iPad Mini to be transformed into a handheld microscope with objective semen analysis software for equine available through the Apple Store (version 4.5.2). The aim of this study was to compare iSperm values for sperm motility and sperm concentration to current acceptable methods for semen analysis and to determine the agreement with these methods using statistical methods. Two ejaculates from each of five Standardbred stallions were used to compare sperm motility (computer-assisted semen analysis [CASA] vs. iSperm) and concentration (NucleoCounter SP-100 [NC] vs. hemocytometer vs. iSperm). Data were analyzed by first testing for the differences between the means of each method using a linear mixed-effects model. The agreement between the two continuous measurements for each method was then investigated by computing Lin's concordance correlation coefficient (CCC), with a value of 1 indicating perfect agreement between methods. Results are reported as the CCC with the associated 95% confidence interval in parentheses. Means for both total motility (TM) and progressive motility (PM) were equal between CASA and iSperm values (P = 0.0741 and P = 0.725, respectively). However, means for all velocity measurements were significantly different between CASA and iSperm readings (P < 0.001). For concentration, means were equal between NC and iSperm values (P = 0.748) and for hemocytometer and iSperm values (P = 0.953). The CCC for TM was 0.871 (0.788, 0.923) and for PM was 0.916 (0.847, 0.955) indicating good agreement between methods. Low levels of agreement were observed for all velocity measurements. Finally, the CCC for concentration compared by iSperm and NC was 0.970 (0.949, 0.982) and for iSperm and hemocytometer it was 0.962 (0.934, 0.978), both close to the line of perfect concordance. Although more work is needed to improve the iSperm software for velocity measurements to be acceptable by research standards, in its present form the iSperm will introduce a low-cost and affordable method for on-farm semen analysis (TM, PM, concentration) for breeders and veterinarians. As a result, more farms will have access to accurate sperm analysis tools which will help to standardize semen processing procedures leading to better overall quality of semen used for artificial insemination.

Using Estrous Behavior to Time Initiation of Oxytocin Administration to Prolong Luteal Function in Mares.

The objective of this study was to use estrous behavior alone to determine the appropriate time for beginning an oxytocin treatment protocol for estrus suppression. We hypothesized that administration of oxytocin beginning 8 days after the onset of estrus will prolong the luteal phase in mares. Twenty-three light breed mares (aged 4-20 years) were exposed to a stallion and observed for signs of sexual receptivity. Mares not displaying signs received 250 µg of cloprostenol intramuscularly (IM) and were teased again 3-4 days later. On the day that estrous behavior was observed (Day 0), mares were randomly divided into two groups: oxytocin (n = 11): oxytocin (60 IU, IM) was administered once daily from Day 8-17; control (n = 12): did not receive treatment. Blood was collected from all mares every 4 days throughout Day 17, and every 7 days thereafter until Day 45. Serum progesterone concentrations >1.0 ng/mL were indicative of a functioning corpus luteum. Interestrus interval was defined as the period between Day 0 and the day when progesterone next reached <1.0 ng/mL. The average interestrus interval was higher for treated mares compared with control mares (32.4 ± 4.2 vs. 21.8 ± 1.5 days, respectively, P = .01). In the oxytocin group, the interestrus interval was longer than 31 days in 6 of 11 (54.5%) mares and up to 45 days in 5 of 11 mares (45.5%). We conclude that luteal maintenance beyond 30 days was attained by once-daily oxytocin administration beginning 8 days following behavioral estrus in a majority of mares.

Whelping and Dystocia: Maximizing Success of Medical Management.

Dystocia can be defined broadly as "difficult birth" or more specifically as difficulty in the bitch expelling the pups through the cervix, vagina, and vestibule. It is a fairly common emergency presented to the small animal practitioner with an incidence rate of approximately 5%. The incidence of dystocia is highest in toy and brachycephalic breeds, and occurs frequently in small litters (<3 pups) due to fetal oversize and delayed onset of labor. As duration of labor progresses, the mortality of the pups and even the bitch increases, with the highest number of stillborn pups occurring after 6 hours from the time of active parturition. Practitioners should understand the mechanism of normal parturition so that abnormalities can be readily identified and medical or surgical intervention performed in a safe and timely manner.

Pregnancies attained after collection and transfer of oocytes from ovaries of five euthanatized mares.

After euthanasia, ovaries were removed from 5 horses and shipped to a laboratory where 46 oocytes were collected. The oocytes were cultured for 24 to 30 hours, and 36 oocytes were transferred to 10 recipient mares via flank laparotomies. Recipient mares were inseminated with semen from various stallions. Sixteen days after transfer, 4 of the recipients were pregnant with at least 1 embryonic vesicle. Embryonic death occurred in 3 recipients, whereas a healthy live foal was born from 1 recipient. Ovaries from valuable mares can be a source of viable oocytes after death of the mare. For shipping to a laboratory, fluctuations in temperature should be minimized and the ovaries should not be chilled.

Effects of milk proteins on sperm binding to the zona pellucida and intracellular Ca(2+) concentration in stallion sperm.

Objectives were to determine the effects of extracellular Ca(2+) and milk proteins on intracellular Ca(2+) concentrations in stallion sperm; and to determine the effects of single caseins on sperm binding to the zona pellucida (ZP). In Experiment I, sperm were incubated in media containing 2 or 4mM Ca(2+) and intracellular Ca(2+) concentration was determined after ionomycin treatment and long-term incubation (3h). Extracellular Ca(2+) concentrations (2 compared with 4mM) did not affect baseline intracellular Ca(2+) concentration of sperm. However, incubating sperm in a medium containing 4 compared with 2mM Ca(2+) resulted in greater (P<0.05) influx of Ca(2+) into sperm. In Experiment II, sperm incubated in media containing 1mg/mL of native phosphocaseinate (NP) or sodium caseinate (SC) showed similar baseline intracellular Ca(2+) and influx of Ca(2+) than control (TALP). In Experiment III, sperm-ZP binding assays were performed in TALP medium containing: no additions (TALP); 1mg/mL SC; 1 or 3mg/mL of α-casein; 1 or 3mg/mL of ß-casein; and 1 or 3mg/mL of κ-casein. The number of stallion sperm bound to bovine ZP was greatest (P<0.05) when SC was used. Co-incubation in media containing single caseins (α-, ß- or κ-casein) resulted in similar results to TALP; however, a dose effect (P<0.05) was observed for ß- and κ-caseins. In conclusion, extracellular Ca(2+) concentration and milk proteins did not affect baseline intracellular calcium in stallion sperm. It appears that ß- and κ-caseins may be responsible for enhancing sperm binding to ZP, but the mechanism remains unknown.

Use of parentage testing to determine optimum insemination time and culture media for oocyte transfer in mares.

Parentage identification was used to test the developmental competence of oocytes cultured under different conditions and fertilized in vivo after oocyte transfer. Oocytes were collected transvaginally from follicles of estrous mares approximately 22 h after administration of human chorionic gonadotropin. Oocytes were cultured for approximately 16 h in one of three media, with or without addition of hormones and growth factors. Groups of three or four oocytes, cultured in different media, were transferred into the oviduct contralateral to a recipient's own ovulation. Recipients were inseminated with semen from two different stallions at 15 h before and 2.5 h after oocyte transfer. Sixteen days after transfer, embryos were recovered from uteri and submitted for parentage testing. The percentage of oocytes resulting in embryonic vesicles was nearly identical (P >0.05) for transferred oocytes (32/44, 73%) versus ovulated oocytes of recipients (9/13, 69%). More (P <0.01) oocytes were fertilized by sperm inseminated before (35/38, 92%) versus after (3/38, 8%) oocyte transfer. Tissue culture medium (TCM)-199 was superior to equine maturation medium I (EMMI; a SOF-based medium) for culturing oocytes (P <0.05), although addition of hormones and growth factors during culture did not improve (P >0.05) development of embryos.