Characterization and cooled storage of semen from corn snakes (Elaphe guttata).

The phylogenetic order Squamata has many representatives that could benefit from the use of semen preservation as a tool for assisting conservation. To date, few studies have been made evaluating the potential for collecting and preserving semen from snakes. The objectives of this study were to characterize semen parameters of the corn snake (Elaphe guttata), including appearance, volume, concentration, sperm motility, and sperm morphology, and to determine the longevity of corn snake sperm motility stored at 4 degrees C. Single semen samples were collected from 22 adult corn snakes. The appearance of the corn snake semen was generally cloudy, and the color was white to tan. Corn snake spermatozoa initially exhibited a median motility of 92.5%. Corn snakes were found to produce small-volume ejaculates (median 0.01 ml). However, the overall concentration of the snake ejaculate was high (chi = 852 x 10(6) +/- 585 x 10(6) spermatozoa/ml). Morphologically, a mean of 75.7 +/- 9.3% of the sperm cells in an ejaculate were normal. Snake ejaculate with a white appearance had significantly higher sperm concentrations (chi = 1,859 x 10(6) +/- 1,008 x 106 sperm cells/ml; F = 15.74, P = 0.001) than tan ejaculates (chi = 601 x 10(6) +/- 439 x 106 sperm cells/ml). Sperm motility decreased significantly in samples that were stored at 4 degrees C for greater than 48 hr in a refrigerator or Equitainer I. This is the first study to characterize semen volume, appearance, and concentration; sperm motility; and sperm morphology in captive corn snakes. The information derived from this study can be used to develop a model for a collection, cooled storage, and shipping program for semen from endangered or threatened captive and wild snakes.     

METASTATIC GRANULOSA CELL TUMOR IN A MANGROVE SNAKE (BOIGA DENDROPHILA MELANOTA)

A 12-year-old, female mangrove snake (Boiga dendrophila melanota) was presented with a 1-month history of anorexia, lethargy, and progressive distention of the caudal coelomic cavity. Based on ultrasonographic and cytological examination, an ovarian neoplasia was considered to be the presumptive antemortem diagnosis. Histological examination confirmed the diagnosis of a granulosa cell tumor (GCT) with metastatic tumors in the coelomic fat bodies. Four months following oophorectomy and surgical removal of the distant metastasis, the snake was euthanized because of sudden onset of lethargy, cardiomegaly, and dyspnea. Postmortem examination revealed the presence of metastatic tumors in the lung, liver, and kidneys. Primary ovarian neoplasms are relatively rare in reptiles. Although GCTs have been documented in snakes, definitive diagnosis is typically determined through postmortem histopathological examination. The present case describes a suitable antemortem diagnostic approach of a malignant GCT in a mangrove snake with distant metastases.     

Influence of constant long days on ejaculate parameters of rabbits reared under natural environment conditions of Mediterranean area

Since rabbit bucks are usually housed under constant long daylight in artificial insemination (AI) centers, the main purpose of this study was to investigate whether constant long day influenced ejaculate parameters of rabbits housed in AI centers in the Spanish Mediterranean area. The study was carried out in Murcia, Spain (37° N). Twenty commercial hybrid male rabbits, aged between 14 and 15 weeks, were randomly allotted to two groups and housed under either natural day length (n=10, ND) or a constant 16-h daylight exposure of 16 h (n=10, CLD). Other management conditions, such as air temperature or reproductive handling, were identical for both groups. Two successive ejaculates were collected twice weekly from every male, and the first one was used to monitor ejaculate characteristics. Measurement of semen production, in terms of ejaculate and semen volume, sperm concentration and total sperm per ejaculate, and sperm quality, in terms of motility index, viability, morphology and acrosome integrity, was assessed in 783 ejaculates collected during 15 months (from October to December). No differences (P>0.05) in either semen production or sperm quality were shown among ejaculates collected from rabbits housed under ND and CLD conditions. A limited influence of season was observed (P<0.01); semen volume and motility index were highest and lowest, respectively, during summer. The increase of air temperature and humidity index (THI) had a significant detrimental effect (P<0.01) on both sperm production and quality parameters with a lag of 6 and 3 weeks, respectively. On the basis of these findings, annual variations of semen production and sperm quality in male rabbits seems more related to THI than to daylight length under conditions of AI management in the Mediterranean area of Spain.     

Relationship between sperm quality traits and field-fertility of porcine semen

An investigation involving seven boars, active in artificial insemination, and 1,350 multiparous sows was conducted at a private farm and aimed at examining the relationship between sperm quality traits and boar fertility in terms of farrowing rate and litter size. This experiment was done for 6 months. The semen samples were evaluated for subjective sperm motility and concentration. Ejaculates with at least 1 × 10⁸ sperm/mL and 70% sperm progressive motility were extended with a commercial medium to 30 × 10⁶ sperm/mL and used for artificial insemination (AI). AI dose was 100 mL semen containing 3 × 10⁹ spermatozoa. Aliquots of diluted semen were assessed for live morphologically normal spermatozoa (LMNS, eosin-nigrosin stain exclusion assay) and sperm chromatin instability (SCI, acridine orange assay). Farrowing rates according to different boar sperm varied (p < 0.001) from 59.3 to 88.92%. The mean values of LMNS (47.2~76.5%) and SCI (0.16~4.67%) differed significantly among boars. LMNS (r = 0.79, p < 0.05) and SCI (r = -0.90, p < 0.02) accounted for 62.2 and 81.7% of the variability in farrowing rates, respectively. After the combination of sperm traits, the relationship between percentage of LMNS with stable chromatin structure and farrowing rate was significant (r = 0.86, p < 0.05). The number of live piglets per parturition was not significantly correlated with sperm quality attributes. In conclusion, boar fertility after AI with freshly diluted semen can be predicted based on the evaluation of sperm morphology and chromatin integrity.     

Reproductive season and male effect on quantitative and qualitative traits of individually collected Muscovy duck (Cairina moschata) semen

Males of Muscovy duck (Cairina moschata) are mainly used for mule duck production via artificial insemination of females originated from wild mallard duck (Anas platyrhynchos); therefore, the quantity and quality of drake semen play a crucial role. The assessment results of male reaction to sexual stimulation by dummy female and basic semen characteristic (ejaculate volume, sperm concentration and morphology) of 12 individually kept Muscovy drakes carried out during the entire reproductive season are described. The male and period of the reproductive season effect on scored semen traits are documented. In total, 792 individual semen collections and evaluations were performed. The average of positive reaction in the entire reproductive season varied from 90.6% in December and April to 50.0% in July, while for individual males, it varied between 97.1% and 29.0%. Throughout the season, the ejaculate volumes ranged from 0.05 to 2.45 ml, sperm concentration from 0.15 × 10⁹ ml⁻¹ to 4.44 × 10⁹ ml⁻¹, total number of live spermatozoa from 68.0% to 100% and live normal (properly formed, with any deformations) from 51.0% to 99.0%. Our study indicates the necessity of male breeders pre-selection before the onset of the reproductive season, and the need to leave an appropriate number of males to ensure adequate amount of semen for female insemination, especially when using Muscovy drakes (Cairina moschata) for interspecies crossing with Anas platyrhynchos ducks.     

Low Dose Insemination in the Sow – A Review

Artificial insemination (AI) in pigs has been established for about four decades but ejaculates are still used insufficiently. Higher demand of semen for AI and new techniques that involve low sperm concentration require the optimization of insemination protocols. Based on the knowledge of the physiology of sperm transportation and events in the female genital tract prior to fertilization, new strategies are under development to minimize sperm losses. One goal is to deposit the semen into the uterine horn rather than into the proximal cervix. It was shown that the minimal number of spermatozoa necessary for surgical AI at the utero‐tubal junction (UTJ) were at least 1 × 10⁶ diluted in 0.5 ml of a special extender. Artificial insemination into the distal part of the uterine horn required about 1 × 10⁷ million sperm in 20 ml of extender. Meanwhile, first insemination devices for non‐surgical intra‐uterine AI are commercially available. Using similar sperm concentrations as for surgical AI, non‐surgical uterine insemination did not differ significantly from control inseminations in terms of pregnancy rate and litter size. With respect to the fertilizing capacities of their ejaculates, boars have to be selected more strictly for sperm quality parameters as most of the compensatory effects of sperm cells disappear in maximally extended semen samples.     

Comparative efficiency of novel laparoscopic and routine vaginal inseminations with cryopreserved semen in rabbits

Laparoscopic artificial insemination technique (LAI) is described to overcome reduced fertility problems in sheep artificial insemination (AI) programmes with frozen semen. Later on, this technology was modified for endangered non-domestic cats to deposit low quality or reduced number of sperm cells hardly obtained by electro-ejaculation into the oviduct. This technique by passes the complex structure of cervix and efficiently transfers the sperm cells to the point of fertilization. In recent years, rabbits are becoming popular transgenic animal models producing various therapeutic and commercial products, as well as being experimental animals for disease models. The worldwide transportation of frozen semen and re-establishment of transgenic lines using AI technology has become a common practice. Therefore, this study was designed to describe a laparoscopic intrauterine insemination technique, which might assist in conceiving the animals with limited number of sperm cells. The female rabbits were laparoscopically (n = 22) or vaginally (n = 13) inseminated with frozen–thawed semen samples containing approximately 10 × 10⁶ motile sperm. The laparoscopic insemination technique provided higher pregnancy rate (45.5%) than vaginal insemination technique (7.7%) (p < .05). In conclusion, the described laparoscopic AI might be a new alternative technique, thus enabling limited or low-quality frozen sperm samples to establish pregnancy in rabbits.     

Influence of Hen Age and Number of Inseminated Sperm on the Number of Holes Hydrolyzed in the Inner Perivitelline Layer of Turkey Eggs

Eggs from young turkey breeder hens have a higher rate of early embryonic mortality (EEM) than eggs from older hens. Preliminary field data indicated that increased sperm concentration decreased the incidence of EEM in eggs from young hens. Possible explanations for decreased EEM following insemination with more concentrated sperm may include altered sperm binding and hydrolyzing of the inner perivitelline layer (IPVL) of eggs from hens of different ages. The current study examined differences in the number of sperm penetration (SP) holes hydrolyzed in the IPVL in hens at 2 different ages when inseminated with 25, 50, 100, 200, 400, or 800 million viable sperm. Hens (12/treatment) were inseminated on d 14 and 21 after photostimulation (32 and 33 wk of age) and were inseminated again at 12 and 13 wk of egg production (44 and 45 wk of age). The SP holes hydrolyzed in the IPVL were counted in the 1,098 eggs produced in the 3 wk following each insemination period. The number of SP holes hydrolyzed in the IPVL was significantly (P ≤ 0.05) greater in younger hens than older hens. Furthermore, the number of SP holes was significantly greater (P ≤ 0.01) with the 400 and 800 million insemination doses as compared with the other 4 insemination doses. There was no interaction between hen age and sperm insemination dose. In conclusion, the absence of an interaction between hen age and insemination dose suggests that factors other than numbers of sperm binding to the IPVL influence EEM. These factors may be a combination of oviduct influences and IPVL properties that change as the hen ages.     

Fertility of Mares Inseminated With Frozen-Thawed Semen Processed by Single Layer Centrifugation Through a Colloid

The aim of this study was to determine whether there was an increase in pregnancy rates when frozen-thawed stallion semen was processed by single layer centrifugation (SLC) through a colloid before insemination. In addition, changes in semen parameters, including motility, were determined before and after SLC. Twenty light-horse mares (aged 3-16 years) and one Thoroughbred stallion (aged 16 years) having average fertility with fresh and cooled semen (>50% per cycle) and displaying a postthaw motility of >35% were used. Control mares were inseminated using 4- × 0.5-mL straws (200 × 10⁶/mL) of frozen-thawed semen. Treatment mares were inseminated with 4 × 0.5 mL of frozen-thawed semen after processing by SLC. Pregnancy rates were compared using Fisher exact test, and continuous parameters were evaluated by a Student t test. The pregnancy rates at day 14 were not different for the mares inseminated with control versus SLC-processed semen, despite the difference in sperm number (171 × 10⁶ ± 21, 59 × 10⁶ ± 25 progressively motile sperm). After frozen-thawed semen was processed by SLC, the percentage progressively motile sperm improved (P < .05), and SLC processing resulted in a 21.8% recovery of spermatozoa. In summary, centrifugation of frozen-thawed semen through a single layer of colloid increased the percentage of motile spermatozoa, but did not improve pregnancy rates after deep horn insemination.     

Economic effects of using sexed semen in Japanese dairy herds

Dairy farm agricultural income should increase when sexed semen is used instead of unsexed semen. This study simulated three scenarios: using unsexed semen (N), using X sperm of Holstein and unsexed semen of Japanese Black (H), and using X sperm of Holstein and Y sperm of Japanese Black (HJ). Two management scenarios were considered: conventional management (CONV); and the use of Japanese Black semen with surplus cows to produce F₁s where sufficient replacement female Holstein calves were obtained for maintenance (MAINT). In CONV, the agricultural incomes of H and HJ were 610 000–2 400 000 yen higher than that of N. The agricultural income increased when the difference in the prices between Holstein males and average F₁ male/female calves was 34 000–50 000 yen, that between Holstein male and female calves was 80 000–110 000 yen, and that between F₁ male and female calves was 50 000–90 000 yen. Therefore, the agricultural income can be increased by using sexed semen to select calves of a more valued breed and sex.     

Laboratory Semen Assessment and Prediction of Fertility: still Utopia?*

Finding a laboratory test reliable enough to predict the potential fertility of a given semen sample or a given sire for artificial insemination (AI) is still considered utopian, as indicated by the modest correlations seen between results obtained in vitro and field fertility. Male fertility is complex, and depends upon a heterogeneous population of spermatozoa interacting at various levels of the female genital tract, the vestments of the oocyte, and the oocyte itself. For this reason, laboratory assessment of semen must include the testing of most sperm attributes relevant for fertilization and embryo development, not only in individual spermatozoa but within a large sperm population as well. Strategies for the discovery of in vitro predictors of semen fertility require evaluations of low sperm doses for AI, so that differences in innate in vivo fertility can be accurately detected.     

Sperm Pre-Incubation Prior to Insemination Affects the Sex Ratio of Bovine Embryos Produced in vitro

The objective of the present study was to determine whether sperm incubation prior to oocyte insemination in vitro affects the sex ratio of resulting blastocyst. Cumulus–oocyte‐complexes (COCs) collected from slaughterhouse ovaries were matured in vitro and inseminated with frozen‐thawed semen of three proven artificial insemination (AI) bulls pre‐incubated in vitro in Sperm‐Talp for 6 and 24 h. On day‐9 blastocysts were collected and processed for sex determination. More than 80% of blastocyst were successfully sexed. There were no significant differences in cleavage and blastocyst rates using sperm pre‐incubated for 6 h as compared with the 0‐h pre‐incubation control group. The cleavage and blastocyst rates were significantly lower in the 24‐h pre‐incubation group. The male to female ratio, when compared with the theoretical 1 : 1, differed significantly in favour of females among hatched (viable) blastocysts derived from sperm pre‐incubated for 24 h prior to insemination as well as among all blastocytsts in the 6‐h group. Moreover, when the sperm treatment was considered, the sex ratio was affected only among hatched blastocysts in 24‐h pre‐incubation group. It was concluded that prolonged sperm pre‐incubation influences the rate of development and the sex ratio among hatched blastocysts.     

Stallion Sperm Integrity After Centrifugation to Reduce Seminal Plasma Concentration and Cool Storage for 4 days

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.     

Case Study of Processing and Insemination Techniques: Attempts to Improve Fertility of an Aged Stallion with Dilute Semen of Poor Quality

The objective of this case study was to investigate whether semen centrifugation and low-dose insemination techniques would improve fertility of an aged subfertile Quarter Horse stallion with low sperm concentration, motility, and morphology in ejaculates. Forty-five mares were bred by one of five treatments (n = 9 per group) using the entire ejaculate as follows: (1) Group Body: body insemination with ejaculate diluted 1:1 in TAMU extender; (2) Group Body-Cent: body insemination after centrifugation and re-suspension of sperm pellet to 1 mL in TAMU extender; (3) Group Horn-Cent: deep horn insemination after centrifugation and re-suspension of sperm pellet to 1 mL in TAMU extender; (4) Group Cent-Hys: hysteroscopic insemination onto the uterotubal papilla after centrifugation and re-suspension of sperm pellet to 200 μL in Kenney-Modified Tyrode’s extender; and (5) Group Dens-Hys: hysteroscopic insemination onto the uterotubal papilla after discontinuous density gradient centrifugation and re-suspension of the sperm pellet in 200-μL Kenney-Modified Tyrode’s extender. Pregnancy rates did not differ among treatment groups (P = .77). Semen centrifugation for low dose insemination did not appear to improve fertility of this subfertile stallion, despite use of entire ejaculates for each individual insemination dose.     

Influence of Steroidal Anti-Inflammatory Drugs on Viability and Fertility of Equine Semen

Considering the efficiency of steroidal anti-inflammatory drugs (SAIDs) in the immunomodulation of breeding-induced endometritis and the possibility of using these drugs by intrauterine route instead of the parenteral application, the present study aimed at evaluating the effect of SAIDs added to the semen extender on equine sperm viability and fertility. In experiment 1, 15 SAIDs were individually added to a skim milk-based extender and, based on the results of sperm motility, dexamethasone was the drug of choice for the subsequent trials. The effect of dexamethasone on the viability of fresh and 24-hour cooled semen was investigated in experiment 2. In experiment 3, fertility rate was measured in both post-breeding endometritis-resistant and susceptible mares. Although dexamethasone supplementation caused a premature decrease in sperm total and progressive motility and in sperm velocities (P < .05), no difference was observed for sperm membrane integrities and fertility (P > .05). Based on these results, we can conclude that dexamethasone can be added to equine semen at the time of insemination or before cooling, although its use was not able to increase fertility.     

Fertility in Holstein × Gyr cows in a subtropical environment after insemination with Gyr sex-sorted semen

Breeding records, including 649 inseminations during fall and winter at a dairy farm in a subtropical area of Western Mexico (24o N; 24°C, mean annual temperature 24°C) were analyzed to document effects of sex-sorted semen from commercial Gyr bullls, estrus synchronization protocol, inseminator, sire and environmental conditions on fertility of crossbred cows (Holstein × Gyr). Percentage of services resulting in pregnancies decreased sharply when sex-sorted semen was used (22.7 vs. 37.7%; P < 0.01). Although statistically not significant (P = 0.31), cows whose first insemination was in November experienced a numerically greater reduction (21 percentage points) in pregnancy rate compared to cows whose first insemination occurred in December. Substantial increases in services per pregnancy (4.71 ± 1.35 vs. 2.13–2.43; P < 0.01) were associated with the warmer month of the study period, November, compared to other winter months. Pregnancy rates of cows regardless of semen category (33%) were not affected by sire, temperature–humidity index and estrus synchronization protocol. Cows inseminated by one inseminator had higher pregnancy rates (P < 0.01) than cows inseminated by other two technicians. The sorted sperm produced 91% (142/156) female offspring. It was concluded that, under the field conditions of the present study, pregnancy rate with sexed semen was 15 percentage points lower than pregnancy rates using conventional semen, with 91% of female calves derived from sexed sperm.     

猫人工授精技术研究进展

人工授精技术是一种较先进的辅助生殖技术,可提高繁殖育种的效益,但因猫类较独特的生殖特点,其人工授精技术发展受限。近年来,科研人员和临床工作者借鉴其他物种相对成熟的人工授精技术,结合猫类生殖特点,对猫人工授精过程中精液采集、精液品质评估、精液保存、刺激母猫排卵方式及具体的人工输精等相关技术进行了优化和创新,如开发了新颖实用的尿导管法采精技术以采集猫精液;在传统精液品质评估的基础上,配合使用荧光染色技术、流式细胞技术及体外受精技术对猫精液品质进行评估;探索不同冷冻液配方及冷冻程序对猫精液保存效果的影响,并尝试使用超快速冷冻法保存公猫遗传物质;研究使用不同种类及不同剂量的激素对诱导母猫发情和排卵效果的差异,以及不同输精部位、方法及时机对母猫妊娠结果的影响等。尽管其中一些新技术与方法已在试验动物中彰显出了其实际应用潜力,但当下依旧还存在操作相对复杂、成本较高、效果低于预期等问题,导致目前猫人工授精技术的应用较局限,不如在犬或其他动物中应用广泛,未来还需更进一步研究和探索才能满足临床实际应用的需要。文章就上述猫人工授精技术中各个方面的研究进展进行了综述,以期为该技术的进一步研究和应用提供参考。     

Preservation of fowl semen in liquid nitrogen: Application to breeding programmes

1. A method of freezing semen of individual males was adapted for use under farm conditions using an automated freezing apparatus.

2. An insemination programme to produce high fertility and hatchability with semen which had been deep frozen for 2 months was devised.

3. Over 90% fertile eggs with a 90% hatch of all eggs set was obtained with frozen and thawed semen over a period from the 2nd to the 12th day after the first of four inseminations. The persistency of fertility was also tested and 93, 86.6 and 30.7% of the eggs were fertile during days 2 to 6, 2 to 8 and 9 to 15 after the last insemination.

4. Corresponding with the high fertility rate, chicks were produced by every hen that was inseminated and from every male whose semen was frozen and stored. The implications for future breeding practices of this successful result are discussed.     

Endometrial Explant Culture to Study the Response of Equine Endometrium to Insemination

Mating‐induced endometritis (MIE) is ubiquitous in the horse after natural mating and artificial insemination with frozen/thawed semen causing the most aggressive response. The majority of mares eliminate MIE 24–48 h after insemination. An endometrial explant culture was tested as a potential in vitro exemplar for sperm‐induced MIE. Endometrial prostaglandin F_2α (PGF_2α) secretion and expression of interleukin‐8 (IL‐8) were used as markers of inflammation. Endometrial explants were cultured from uteri collected from follicular phase mares. Explants were challenged with 1 or 10 × 10⁶ sperm/ml frozen/thawed semen, chilled semen, washed sperm or seminal plasma. Medium was collected 24 and 72 h after challenge and assayed for PGF_2α by radioimmunoassay. Treatment of endometrial explants with frozen/thawed, chilled semen or washed sperm did not change the secretion of PGF_2α compared with untreated controls. However, 24 h after challenge cultured explants expressed IL‐8. The in vitro endometrial explant system did not represent the in vivo response to semen when PGF_2α was used as a marker of inflammation, yet the use of gene expression as an inflammatory marker warrants further investigation.