The objective of the study was to investigate if reducing the seminal plasma of stallion extended semen by centrifugation once will suffice to maintain acceptable semen quality for insemination after 4 days of cool storage. Collected semen was extended to 25 × 106 sperm/mL and subjected to one of the following treatments: noncentrifuged (control), centrifuged for 10 minutes at 900 × g and 1800 × g. The supernatant was partially removed, and the sperm pellet, reconstituted and re-extended. It was then placed in a passive cooling device overnight and then transferred to a refrigerator for the remainder of the cooling period. At day 0, 2, and 4, total motility (TM), progressive motility (PM), and plasma (PLM) and acrosomal membrane integrity were assessed. Centrifuged groups had higher TM and PM at day 4 than the control group (P < .05). Likewise, centrifuged groups had higher intact PLM in day 4 (P < .05). A single centrifugation cycle to reduce seminal plasma concentration will suffice to preserve sperm integrity acceptable for an artificial insemination dose up to 4 days of cool storage.

To estimate when, during stallions' extra-gonadal reserves (EGR) depletion period, sperm quality would reach its highest quality, six light breed sexually rested stallions were collected daily for 7 days to deplete EGR. On collection days 1, 3, 5, and 7, sperm output, total (TM) and progressive (PM) motility, morphology, and plasma membrane (PLM) integrity were evaluated. Sperm output decreased as EGR depletion advanced, stabilizing on days 5-7. Sperm motility (TM and PM) and morphology were not different during EGR depletion. Plasma membrane integrity improved from day 1 to 3; however, no further improvement observed on days 5 and 7. Sperm of sexually rested stallions reach the highest quality on day 3 of the EGR depletion period.

It was hypothesized that optimal sperm recovery rate (RR) without damage to the sperm would be obtained after centrifugation without a cushion solution. Semen collected three times from six light breed stallions was extended to 25 × 10(6) sperm/mL and centrifuged at CON (noncentrifuged), 900NC (no-cushion), 900C (cushion), 1800NC, and 1800C × g for 10 minutes. Sperm concentration, motility (TM and PM), and intact plasma membranes (PLM) and acrosomes (ACR) pre- and postcentrifugation (D0) and after 24 hours (D1) of cooling were evaluated. The RR in the CON (100 ± 0.0), 900NC (93.7 ± 2.9), and 1800NC (96.7 ± 2.6) groups was significantly higher than the 900C (68.7 ± 4.6) and 1800C (79.6 ± 3.5) groups. The D0 TM and PM were not different between the CON, 900NC, 900C, and 1800C, but were lower for the 1800NC group. The D1 TM and PM of the 900NC (75.2 ± 3.8 and 71.1 ± 4.1) and 900C (76.2 ± 3.7 and 72.4 ± 4.0) groups were significantly higher than the 1800NC (71.7 ± 4.1 and 67.3 ± 4.4) and 1800C (71.6 ± 4.1 and 67.2 ± 4.4) groups, and the CON (66.2 ± 4.5 and 60.0 ± 4.8) group was significantly lower than the other groups. The D1 PLM of the CON, 900NC, 900C, 1800NC, and 1800C groups were not different. The ACR on D1 was significantly lower for the CON (93.0 ± 2.4) group compared with all other groups. Optimal RR preserving sperm integrity was obtained in the 900NC group.

Persistent organochlorine pollutants such as polychlorinated biphenyls (PCBs), dichlorodiphenyldichloroethylene (p,p'-DDE), and polybrominated diphenyl ethers (PBDEs) are stable, bioaccumulative, and widely found in the environment, wildlife, and the human population. To explore the hypothesis that reproduction in male fish is associated with environmental exposures in the lower Columbia River (LCR), reproductive and endocrine parameters were studied in male resident, non-anadromous largescale sucker (Catostomus macrocheilus) (LSS) in the same habitats as anadromous salmonids having conservation status. Testes, thyroid tissue and plasma collected in 2010 from Longview (LV), Columbia City (CC), and Skamania (SK; reference) were studied. Sperm morphologies and thyrocyte heights were measured by light microscopy, sperm motilities by computer-assisted sperm motion analysis, sperm adenosine triphosphate (ATP) with luciferase, and plasma vitellogenin (VTG), thyroxine (T4), and triiodothyronine (T3) by immunoassay. Sperm apoptosis, viability, mitochondrial membrane potential, nuclear DNA fragmentation, and reproductive stage were measured by flow cytometry. Sperm quality parameters (except counts) and VTG were significantly different among sites, with correlations between VTG and 7 sperm parameters. Thyrocyte heights, T4, T3, gonadosomatic index and Fulton's condition factor differed among sites, but not significantly. Sperm quality was significantly lower and VTG higher where liver contaminants and water estrogen equivalents were highest (LV site). Total PCBs (specifically PCB-138, -146, -151, -170, -174, -177, -180, -183, -187, -194, and -206) and total PBDEs (specifically BDE-47, -100, -153, and -154) were negatively correlated with sperm motility. PCB-206 and BDE-154 were positively correlated with DNA fragmentation, and pentachloroanisole and VTG were positively correlated with sperm apoptosis and negatively correlated with ATP. BDE-99 was positively correlated with sperm counts and motility; T4 was negatively correlated with counts and positively correlated with motility, thus indicating possible androgenic mechanisms and thyroid endocrine disruption. Male LSS proved to be an informative model for studying reproductive and endocrine biomarkers in the LCR.

Two commercially available egg yolk-based semen extenders, one marketed for human semen freezing (HEYE) and one marketed for canine semen freezing (CEYE), were used to cryopreserve semen from single ejaculates of 11 different dogs. For each extender, a 30- and a 60-min cooldown period was used prior to the addition of the extender containing glycerol and then immediately frozen in liquid nitrogen vapours. Sperm motility was measured using a computer-assisted semen analysis (CASA) system. Sperm intact membranes were measured using SYBER-14 and propidium iodide. Semen in the HEYE cooled for 60 min had a significantly greater percentage of intact membranes than the semen in the HEYE cooled for 30 min (p = 0.02). Semen in the HEYE cooled for 60 min had significantly greater total motility (p = 0.007) and progressive motility (p = 0.004) than semen cooled for 60 min in the CEYE and semen cooled for 30 min in the HEYE (total motility p = 0.02 and progressive motility p = 0.02). Semen cooled for 60 min in the CEYE did not differ significantly in total (p = 0.6) or progressive motility (p = 0.4) than semen cooled for 30 min in the CEYE. There was no difference in total (p = 0.8) or progressive motility (p = 0.8) between the semen cooled for 30 min in the HEYE and the semen cooled for 30 min in the CEYE.

Conventional centrifugation protocols result in important sperm losses during removal of the supernatant. In this study, the effect of centrifugation force (400 or 900 × g), duration (5 or 10 min), and column height (20 or 40 mL; Experiment 1); sperm concentration (25, 50, and 100 × 10(6)/mL; Experiment 2), and centrifugation medium (EZ-Mixin CST [Animal Reproduction Systems, Chino, CA, USA], INRA96 [IMV Technologies, Maple Grove, MN, USA], or VMDZ [Partnar Animal Health, Port Huron, MI, USA]; Experiment 3) on sperm recovery and survival after centrifugation and cooling and storage were evaluated. Overall, sperm survival was not affected by the combination of centrifugation protocol and cooling. Total sperm yield was highest after centrifugation for 10 min at 400 × g in 20-mL columns (95.6 ± 5%, mean ± SD) or 900 × g in 20-mL (99.2 ± 0.8%) or 40-mL (91.4 ± 4.5%) columns, and at 900 × g for 5 min in 20-mL columns (93.8 ± 8.9%; P < 0.0001). Total (TMY) and progressively motile sperm yield followed a similar pattern (P < 0.0001). Sperm yields were not significantly different among samples centrifuged at various sperm concentrations. However, centrifugation at 100 × 10(6)/mL resulted in significantly lower total sperm yield (83.8 ± 10.7%) and TMY (81.7 ± 6.8%) compared with noncentrifuged semen. Centrifugation in VMDZ resulted in significantly lower TMY (69.3 ± 22.6%), progressively motile sperm yield (63.5 ± 18.2%), viable yield (60.9 ± 36.5%), and survival of progressively motile sperm after cooling (21 ± 10.8%) compared with noncentrifuged semen. In conclusion, centrifuging volumes of ≤ 20 mL minimized sperm losses with conventional protocols. With 40-mL columns, it may be recommended to increase the centrifugal force to 900 × g for 10 min and dilute the semen to a sperm concentration of 25 to 50 × 10(6)/mL in a milk- or fractionated milk-based medium. The semen extender VMDZ did not seem well suited for centrifugation of equine semen.

ContentsLow‐dose insemination has been proposed to reduce persistent breeding‐induced endometritis (PBIE) in mares with delayed uterine clearance (DUC). Others proposed that hysteroscopic insemination induces an exaggerated inflammatory response and should be avoided in DUC mares. The objectives here were to evaluate presence and severity of PBIE in normal and DUC mares after hysteroscopic insemination with fresh semen, and to determine if hysteroscopy could be used in DUC mares without inducing excessive inflammation. Reproductively normal (n = 4) and DUC (n = 5) mares received four treatments in random order: uterine body insemination (UB, 1 × 109 spermatozoa, 20 ml), hysteroscopic insemination (HYST, 5 × 106 spermatozoa, 0.5 ml), sham hysteroscopic insemination (SHAM, semen extender, 0.5 ml) and hysteroscopic infusion of seminal plasma (SP, 0.5 ml). Significantly more DUC (50%) mares than normal (14%) mares accumulated intrauterine fluid 24 h post‐treatment. The difference in fluid accumulation between DUC (40%) mares and normal (7%) mares was also significant 48 h post‐treatment. Fluid scores were not significantly different between treatments in normal mares. However, treatments HYST and SHAM resulted in significantly higher fluid scores 24 h but not 48 h post‐treatment in DUC mares. There was no effect of treatment or mare group on the percentage and total number of neutrophils in uterine fluid 48 h post‐treatment. Percentage of neutrophils was correlated with duration of hysteroscopy in normal mares, with procedures lasting ≥9 min associated with PBIE. There was no effect of mare group, treatment or duration of hysteroscopy on pregnancy rate. Hysteroscopy induces a transient inflammation that is not more severe than that after conventional artificial insemination, suggesting no contraindication to its use in DUC mares.

Embryo collection in the bitch is usually performed via surgical collection or uterine excision. Our objective was to collect embryos nonsurgically, or with minimal surgical invasion. Estrus was monitored using vaginal cytology and progesterone analysis during 1 natural cycle and 13 cycles induced by the submucosal insertion of a 2.1-mg deslorelin implant into the vestibulum of Walker-type hounds (n = 10, 30 kg). Bitches (n = 14 cycles) were transcervically inseminated twice in 12 cycles, (3.3 and 5.2 days post-LH) and once on 2 cycles (Day 6 post-LH) with fresh semen. Uterine flushes (n = 14) using an equine medium (ViGro, Bioniche Animal Health USA Inc., Athens, GA, USA) were done 13 to 15 days after the LH peak. Nonsurgical, nonanesthetized flushes were attempted in 9 cycles. In one cycle an 8-Fr, 55-cm Foley catheter with a 1-mL cuff (SurgiVet, Waukesha, WI, USA) stiffened with a metal stylette was inserted through the cervix using a 22-Fr sheath (63027KL, Karl Storz Veterinary Endoscopy, Goleta, CA, USA) and a 3.5-mm cystoscope, (63325BA, Karl Storz Veterinary Endoscopy) and flushed with 1.5 mL of medium infused and recovered by aspiration. In a one bitch, an 8-Fr polypropylene catheter (Sovereign, Tyco Healthcare, Mansfield, MA, USA) was transcervically passed after failing to pass an 8-Fr Foley catheter, but a flush was not done. In 5 cycles, the 22-Fr sheath could not be passed into the cranial vagina, so a 9.5-Fr, 43-cm cystoscope (27012L, Karl Storz Veterinary Endoscopy) was used to transcervically pass a 5-Fr, non-cuffed catheter (17500/0005, Minitube of America, Verona, WI, USA). Only non-cuffed catheters could be passed through the 9.5-Fr cystoscope, so no flushes were attempted. In 2 cycles, the cervix could not be visualized. Surgical flushes under anesthesia were attempted in 5 cycles. One was by transcervical catheterization using an 8-Fr catheter identical to the nonsurgical attempts, but adding surgical exteriorization of the uterine horns and retrograde flushing with 20 mL of medium. In 4 bitches the uterus was exteriorized by ventral midline laparotomy; one had a pyometra at surgery, and in three, 14-gauge i.v. catheters (Angiocath, BD, Sandy, UT, USA) were placed in the tip and body of each horn, and the uterus was retrograde flushed with 20 mL of medium. No embryos were recovered by the nonsurgical and/or transcervical flushes. Passing the 22-Fr sheathed cystoscope that accommodated a cuffed catheter into the cranial vagina, passing a cuffed catheter, or visualizing the cervix was unsuccessful in 7 of 9 cycles. Uterine distension appeared to cause discomfort during nonsurgical flushes. Three of 5 bitches that had a 5-Fr, non-cuffed catheter were subsequently pregnant. One blastocyst was recovered after surgical flushing with the i.v. catheters in the uterus. Nonsurgical embryo recovery in the bitch was most likely unsuccessful, compared to ours and previous work, due to inabilities to pass a cystoscope into the cranial vagina, to pass a catheter through the cervix, to visualize the cervix, or to create uterine distension using a small volume of medium. Pregnancies were maintained after successful transcervical catheterization of mated bitches.

The objectives of this study were to determine the effects of centrifugation on equine sperm total and progressive motility, viability, and acrosomal integrity. We hypothesized that although high centrifugation forces would be detrimental to equine Equus caballus sperm, recovery rates would increase. Ejaculates from six stallions were collected, extended to a concentration of 25x10(6) cells/mL, and subjected for 10min to (1) no centrifugation (NC) or (2) centrifugation at 400xg, (3) 900xg, or (4) 4500xg. Before and after centrifugation (Day 0), and after 24h of cooling (Day 1), sperm motility was assessed by computer-assisted semen analysis, and samples were stained with SYBR-14/propidium iodide (PI) for viability and with PI/fluorescein isothiocyanate (FITC)-Peanut aglutinin (PNA) (Arachis hypogaea) for acrosomal integrity. The effect of treatment and day on motility, viability, and acrosomal integrity was determined using a mixed linear model. Compared with the other treatments, centrifugation at 4500xg reduced all end points measured (P<0.05). Both 400xg and 900xg yielded lower recovery rates than that of 4500xg (NC=100.0+/-0.0%; 400xg=54.4+/-8.6%; 900xg=75.0+/-7.1%; 4500xg=97.9+/-2.8%; P<0.05). Centrifugation at 400xg or 900xg did not damage equine sperm. Based on these findings, further studies of centrifugal forces between 900xg and 4500xg are warranted to determine the optimal force that maximizes recovery rate, minimizes sperm damage, and does not affect fertility.

Evaluation of canine cryopreserved semen has the ultimate goal of determining if an individual frozen ejaculate will have acceptable fertility. This is difficult in that there is no accepted normal fertility for the dog. The fertility of the female also plays a crucial role in estimating the fertility of the male. Poor female fertility can make a fertile male appear less fertile. Variability of animals, breeding technique, breeding timing, and number of cells inseminated make comparisons in canine fertility difficult to truly measure. Many more animals are needed to provide meaningful statistical results than are usually used. Several tests, including motility in bright field and phase contrast microscopy, computer analysis of motility, sperm morphology, sperm membrane integrity, capacitation and sperm function tests have been investigated to predict fertility, however few of these tests have actually been correlated with fertility. More work is needed to create one or more tests that accurately predict fertility of cryopreserved canine semen.

A retrospective analysis was performed to determine the effects of age, breed, parity, and litter size on the duration of gestation in the bitch. Bitches at two locations were monitored from breeding to whelping. A total of 764 litters whelped from 308 bitches (36 large hounds, 34 Golden Retrievers, 23 German Shepherd Dogs (GSD), and 215 Labrador Retrievers). By breed, the number of whelpings was 152, 72, 58, and 482 for the hounds, Golden Retrievers, German Shepherd Dogs, and Labrador Retrievers, respectively. Whelping was predicted to be 57 d from the first day of cytologic diestrus in the hounds or 65 d from the initial progesterone rise in the other breeds. The average gestation duration (calculated as 8 d prior to Day 1 of cytologic diestrus in hounds or measured from the initial progesterone rise in other breeds) by breed (days ± S.D.) was 66.0 ± 2.8, 64.7 ± 1.5, 63.6 ± 2.1, and 62.9 ± 1.3 for the hounds, Golden Retrievers, German Shepherd Dogs, and Labrador Retrievers, respectively. The relationship of age, breed, parity, and litter size with the difference in gestation duration was evaluated using log linear modeling. Age or parity had no effect on gestation duration. Compared to Labrador Retrievers, the German Shepherd Dogs, Golden Retrievers and hounds were more likely to have a longer gestation duration; three, four and nearly eight times as likely, respectively. Bitches whelping four or fewer pups were significantly more likely to have a longer gestation duration than those whelping five or more pups; the prolongation averaging 1 d.