Pregnancies from imipramine and xylazine-induced ex copula ejaculation in a disabled stallion.

Breeding or semen collection was attempted using: natural cover, manual stimulation, artificial vagina, pharmacologic induction of ejaculation, and electroejaculation. Sperm cells were recovered from the ductus deferens and epididymides post mortem. Only semen collected ex copula by imipramine and xylazine treatment resulted in conceptions (4/5). This is the first report of pregnancies in horses from ex copula semen collection.     

Effect of seminal plasma concentration and various extenders on postthaw motility and glass wool-Sephadex filtration of cryopreserved stallion semen

OBJECTIVE: To compare the effect of semen extender and seminal plasma on postthaw motility and filtration through a glass wool-Sephadex (GWS) filter for frozen stallion semen. SAMPLE POPULATION: 7 stallions from which we collected > or = 3 ejaculates/stallion. PROCEDURES: 4 experiments were conducted to evaluate postthaw quality of frozen stallion semen. Kenney extender was compared with glucose-EDTA extender by use of various dilution rates that resulted in differing concentrations of seminal plasma. Stallions known to produce semen with poor postthaw quality were used to investigate whether a particular extender or dilution rate could improve ability of such semen to survive freeze-thaw procedures. RESULTS: Use of Kenney extender as the centrifugation extender significantly improved postthaw motility and GWS filtration, compared with glucose-EDTA. Extending semen at a dilution of 1:3 was significantly better than 1:1 for both motility and GWS filtration. In addition, including seminal plasma at a concentration of 5% in the cryopreserved semen resulted in significantly higher yield of spermatozoa after GWS filtration, compared with complete removal of SP or use of seminal plasma at 25%. Lastly, semen with poor postthaw quality had significantly improved postthaw quality in regard to motility and GWS filtration when semen was frozen with seminal plasma at a concentration of 5%, compared with semen frozen with seminal plasma at a concentration of 25%. CONCLUSIONS AND CLINICAL RELEVANCE: Use of Kenney extender at a high dilution (> or = 1:3) immediately after collection of semen can improve postthaw quality of frozen stallion semen.     

Imaging flow cytometry to characterize the relationship between abnormal sperm morphologies and reactive oxygen species in stallion sperm

Low levels of intracellular reactive oxygen species (ROS) are essential for normal sperm function and are produced by sperm mitochondria as a byproduct of metabolism, but in excess, ROS can cause catastrophic cellular damage and has been correlated with infertility, poor sperm motility and abnormal morphology in humans. Stallion sperm motility is fueled predominantly by oxidative phosphorylation-produced ATP, requiring high basal rates of mitochondrial function. Consequently, whether elevated ROS production by stallion sperm is an indicator of dysfunctional or highly motile cells has been debated by researchers over the last decade. The objective of this study was to evaluate the relationship between various sperm morphologies and ROS production in fresh and cooled stallion semen by employing the novel method of imaging flow cytometry for stallion semen assessment. For evaluation of fresh semen, single ejaculates (n = 5) were collected from four resident stallions at the University of California, Davis. For the evaluation of 24-h cool-stored semen, single ejaculates were collected from stallions at Texas A&M University (n = 5) and shipped to the University of California, Davis overnight for evaluation. Ejaculate volume, sperm concentration and motility parameters were recorded. Samples were co-stained for viability and ROS detection with SytoxGreen™ and dihydroethidium (DHE), respectively, and evaluated with the Amnis® ImageStream® system (Luminex Corporation). Antimycin, an electron transport chain inhibitor that triggers ROS production (1 μM), was used as a positive control for DHE, while dead cells (2× snap frozen in liquid nitrogen) served as a positive control for SytoxGreen™. Unstained samples were also evaluated as controls. Imaging flow cytometric analysis was performed with the ideas ® software (Luminex Corporation). Evaluated morphologies included abnormal head (AH), abnormal midpiece (AM), abnormal tail (AT), proximal cytoplasmic droplet (PD), or distal cytoplasmic droplet (DD), and morphologically normal (MN) cells. For fresh semen, an additional abnormality, coiled tail and midpiece (CTM) was assessed; 24-h cool-stored semen did not contain enough viable CTM cells for analysis. Only cells with obvious, single abnormalities were selected for the first portion of analysis to minimize subjectivity. Mixed effects modelling was used to evaluate the relationship between each morphologic classification and the corresponding DHE fluorescence intensity. Compared to the MN population, ROS production was significantly higher in viable cells with AH, PD and AM (p < .0001) in both fresh and cooled semen. CTM cells had significantly higher levels of ROS production compared to MN cells in fresh semen (p < .0001). There was no significant difference in ROS levels between MN cells and AT and DD cells in either fresh or cooled semen (p > .05). These results suggest that ROS generation is indicative of abnormal cell morphology and function and confirm that imaging flow cytometry is a valuable tool for the assessment of stallion semen.     

Comparison of pregnancy per AI of heifers inseminated with sex-sorted or conventional semen after oestrus detection or timed artificial insemination

The objective of the study was to compare the fertility after using sex-sorted or conventional semen either with oestrus detection (EST) or timed artificial insemination (TAI) in Holstein heifers. Holstein heifers were randomly assigned to one of the following treatments in a 2 × 2 factorial design. Heifers in the EST group were inseminated with sex-sorted (n = 114) or conventional semen (n = 100) after spontaneous or induced oestrus. Heifers in the TAI, subjected to the 5-day Cosynch+Progesterone protocol (GnRH+P4 insertion-5d-PGF_2α+P4 removal-1d-PGF_2α-2d-GnRH+TAI), were inseminated with sex-sorted (n = 113) or conventional semen (n = 88). Statistical analyses were performed using PROC GLIMMIX procedure of SAS 9.4 (SAS Institute Inc., Cary, NC). Overall P/AI was 60.7% for EST and 54.2% for TAI regardless of types of semen and 68.1% for conventional and 48.9% for sex-sorted semen regardless of insemination strategies. Fertility of heifers inseminated with either sex-sorted (53.5%; 44.2%) or conventional (69.0%; 67.0%) semen did not differ between EST and TAI respectively. Besides, the interaction between the semen type and the insemination strategy was not significant for P/AI. The embryonic loss was significantly greater with sex-sorted semen (17.1%) compared to conventional semen (1.6%). There was no sire effect with sex-sorted semen on P/AI (52.6% vs. 46.2%) and embryonic loss (16.4% vs. 18.0%). As expected, sex-sorted semen resulted in more female calves (89.8% vs. 51.6%) than conventional semen. Thus, sex-sorted semen can be used with 5-day Cosynch+Progesterone protocol to eliminate the inadequate oestrus detection and to increase female calves born in dairy heifers.     

Application of Techniques for Sperm Selection in Fresh and Frozen-Thawed Stallion Semen

The objective of this research was to improve the techniques in processing chilled and frozen‐thawed horse semen. In a preliminary experiment (Exp. I), different techniques for sperm selection and preparation [Swim‐up, Glass wool (GW) filtration, Glass wool Sephadex (GWS) filtration; Percoll] were tested for their suitability for equine spermatozoa and results were compared with the routine procedure by dilution (Exp. I). In the main experiment (Exp. II), two sperm preparation techniques (GWS, Leucosorb^®) refering to the results of Exp. I and a previous study of our group (Pferdcheilkunde 1996 12, 773) were selected for processing complete ejaculates either for cooled‐storage or cryopreservation. In a third experiment (Exp. III), pregnancy rates from inseminations with semen processed according to the techniques tested in Exp. II were compared with those obtained with semen processed according to routine procedures. In Exp. I (six stallions, six ejaculates/stallion), between 48 and 92% of spermatozoa were lost following the different sperm selection procedures (p < 0.05). Preparation of sperm increased percentage of progressively motile spermatozoa (pms) [Swim‐up, GW, GWS vs dilution, Percoll (p < 0.05)] and decreased percentage of sperm head abnormalities [Swim‐up, GW, GWS vs dilution, Percoll (p < 0.05)] probably by not improving the quality of individual cells, but by elimination of spermatozoa of inferior quality. In Exp. II (eight stallions, three ejaculates/stallion) Leucosorb^® and GWS procedures allowed the filtration of large volumes (extended ejaculates) for routine laboratory practice. GWS and Leucosorb^® filtration resulted in increased motility, membrane integrity and sperm viability after storage of spermatozoa until 48 h at +5°C when compared with control (diluted) and centrifuged semen (p < 0.05). Significantly more spermatozoa were recovered after centrifugation (87.8 ± 15.4%) compared with GWS (63.5 ± 18.6%) and Leucosorb^® filtration (53.6 ± 22.3%). GWS or Leucosorb^® procedure resulted in successful cryopreservation of stallion semen without centrifugation for removal of seminal plasma. The per cycle conception rate of inseminated mares using 200 × 10⁶ pms transferred within 8 h after collection of semen was not affected by GWS filtration or Leucosorb^® separation when compared with centrifugation (n.s.; Exp. III). In conclusion, GWS and Leucosorb^® filtration results in the improvement of semen quality and should be considered as a method for stallion semen processing. Additional studies are needed for the evaluation of potentially higher fertilizing ability of stallion spermatozoa separated by techniques for sperm selection.     

Effects of Brucella ovis infection on semen characteristics of 16-month-old red deer stags

AIM: To determine the effects of Brucella ovis infection on semen characteristics of 16-month-old red deer stags (Cervus elaphus). METHODS: At monthly intervals during March, April and May, semen was collected using electro-ejaculation from 9 yearling red deer stags that had been artificially infected with B. ovis 3 months previously. In March, semen was also collected from 6 non-infected stags from the same peer group for comparison. Semen was evaluated for gross appearance, percentage of sperm showing forward motility, sperm morphology and sperm density/ml of semen, and the presence of white blood cells was determined. In addition, at the time of semen collection, the epididymes of each stag were palpated and lesions recorded. RESULTS: Grossly visible purulent material was present in semen from 6/9 infected stags and the percentage of sperm showing forward motility did not exceed 20% in any of these samples. Increased numbers of white blood cells and cellular debris were evident in semen from 8/9 infected stags. Compared with non-infected stags, sperm motility in semen from infected stags was significantly reduced (p<0.05). There were no differences in percentage of morphologically normal sperm between groups, but infected stags had more detached sperm heads present in semen than non-infected stags (p<0.05). Only 2 of the infected stags had lesions of epididymitis evident on scrotal palpation. CONCLUSIONS: B. ovis infection is likely to have a detrimental effect on stag semen quality. While there was variation between individual animals, overall sperm motility was decreased and semen from infected stags had increased numbers of white blood cells and detached sperm heads. CLINICAL RELEVANCE: The fertility of breeding stags may be reduced if they are infected with B. ovis and this possibility should be considered when investigating reproductive problems of deer.     

Single and double layer centrifugation improve the quality of cryopreserved bovine sperm from poor quality ejaculates

Background: Density gradient centrifugation was reported as a technique of semen preparation in assisted reproductive techniques in humans and animals. This technique was found to be efficient in improving semen quality after harmful techniques such as cryopreservation. Recently a modified technique, single layer centrifugation,was proposed as a technique providing a large amount of high quality spermatozoa, and this treatment was performed before conservation. Single layer centrifugation has been studied prevalently in stallions and in boars,but limited data were available for bulls. Occasionally bulls are known to experience a transient reduction in semen quality, thus techniques that allow improvement in semen quality could be applied in this context. The aim of this study was the evaluation of single layer and double layer centrifugation by the use of iodixanol, compared with conventional centrifugation and non-centrifuged semen, on the sperm characteristics during the cryopreservation process in bulls with normal and poor semen quality.Results: Single layer centrifugation and double layer centrifugation both significantly increased the percentage of normal spermatozoa and decreased the percentage of non-sperm cells in poor quality samples, while both were ineffective in those of normal quality. Sperm characteristics in poor quality samples increased after single layer centrifugation and double layer centrifugation, reaching values similar to those recorded in normal samples, and this trend is maintained after equilibration and after cryopreservation. On the other hand, SLC and DLC resulted in a consistent reduction in the spermatozoa recovered, and this resulted in a reduction of the absolute amount of spermatozoa cryopreserved in the normal samples, without a clear improvement in sperm characteristics in this type of sample.Conclusions: These data suggested that both SLC and DLC could be performed in practice, but their application should be limited to the cases in which the quality of the spermatozoa recovered is more important than the total amount of spermatozoa.     

Conception rates in European fallow does (Dama dama dama) following intrauterine insemination with frozen-thawed semen from Mesopotamian fallow (Dama dama mesopotamica) and crossbred (Dama dama dama x Dama dama mesopotamica) bucks

Ninety eight parous fallow does received laparoscopic intrauterine insemination of frozen-thawed semen at one of 2 fixed intervals following oestrus synchronisation treatment. Semen was collected from a Mesopotamian (Dama dama mesopotamica) and a crossbred (F1) (Dama dama dama x Dama dama mesopotamica) fallow buck. Does were inseminated at either 56 or 66 hours after the removal of an intravaginal controlled internal drug releasing device. Eighty eight does received a single straw of frozen-thawed semen containing a total of 50 x 10(6) spermatozoa, while the remaining 10 received split straws containing 25 x 10(6) spermatozoa. Overall, the use of F1 semen containing 50 x 10(6) spermatozoa resulted in a 68% (17/25) conception rate compared with the Mesopotamian semen, which resulted in a 41% (26/63) conception rate. Conceptions were also achieved using 25 x 10(6) spermatozoa of either Mesopotamian or F1 semen (3/8 versus 2/2, respectively). Overall, the conception rate was higher for F1 than Mesopotamian semen (P less than 0.025) and there was a significant interaction with time of insemination (P less than 0.05); for F1 semen there was no difference in conception rate at the 2 insemination times, but for Mesopotamian semen conception was significantly higher (P less than 0.005) following insemination at 66 hours than at 56 hours.     

Myeloperoxidase in Equine Semen: Concentration and Localization during Freezing Processing

Myeloperoxidase (MPO) is a pro-oxidant enzyme contained in and released by neutrophils during degranulation or after lysis. Post-thaw semen contains MPO, and concentration of this enzyme is associated with decreased motility. The aim of this study was to determine kinetics of MPO concentration during freezing, its origin, and its impact on frozen-thawed semen. Forty ejaculates were used. Semen was frozen using the classical freezing procedure. MPO concentrations were assayed in fresh semen, after centrifugation, and after cooling down to 4°C. Post-thaw MPO assay results and spermogram characteristics were determined. MPO immunocytochemistry was performed on 4 different ejaculates at each step of freezing procedure. MPO concentration increased after cooling down to 4°C and thawing compared with fresh semen. As temperature decreased, MPO was higher or tended to be higher in post-thaw poor quality samples. Nonsperm cells showed various degrees of MPO immunostaining and were observed as epithelial cells with nuclear pyknosis and keratinization. MPO immunostaining increased in medium and decreased in nonsperm cells during freezing. Our study shows that MPO concentration in equine semen increases when temperature decreases. We hypothesize that nonsperm cells present in fresh semen could release MPO.     

FERTILITY OF FROZEN BOAR SEMEN USED FOR AI IN COMMERCIAL SETTINGS

Surgical insemination was the least efficient means to procure offspring (27% farrow rate). However, it offers to be an effective means for generating offspring from limited semen supplies or semen having high value (i.e. genetic merit or gender selected sperm). Transcervical AI resulted in an overall 40% farrow rate. However, there was a significant farm effect observed and this was anticipated in considering the variation in management and personnel practices.
The application of frozen boar semen for commercial practice has merit. Although the fertility of frozen semen is below that of fresh semen, it is a low cost and efficient method for introgressing new germplasm (genetics) into a current genetic base. With respect to seedstock suppliers, the lowered fertility associated with frozen semen is acceptable when it is looked upon as a means to introduce new genetics for research purposes.     

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.     

Unusual ovarian activity in a mare preceding the development of an ovarian granulosa cell tumour

An 8-year-old mare, with a foal at foot, was inseminated on foal heat with frozen semen, with the resultant pregnancy lost between days 34 and 41. The right ovary developed a large anovulatory follicle that was non-responsive to multiple doses of ovulating agents. The follicle eventually appeared to luteinise, although plasma progesterone concentrations did not reflect this. Another follicle developed, responded to GnRH and resulted in a pregnancy from frozen semen that went to term with a healthy foal. When the mare was examined after foaling, the structure on the right ovary appeared to be a granulosa cell tumour; the left ovary was smaller than normal and non-functional. Surgical removal of the right ovary before increasing photoperiod resulted in a return to function of the left ovary and a pregnancy to frozen semen on the second cycle following removal. Figures showing concentrations of inhibin, progesterone, androstenedione, oestradiol and testosterone are presented for this entire period. Unusual ovarian activity in the mare might be a prelude to the development of a granulosa cell tumour.     

Unusual ovarian activity in a mare preceding the development of an ovarian granulosa cell tumour

An 8-year-old mare, with a foal at foot, was inseminated on foal heat with frozen semen, with the resultant pregnancy lost between days 34 and 41. The right ovary developed a large anovulatory follicle that was non-responsive to multiple doses of ovulating agents. The follicle eventually appeared to luteinise, although plasma progesterone concentrations did not reflect this. Another follicle developed, responded to GnRH and resulted in a pregnancy from frozen semen that went to term with a healthy foal. When the mare was examined after foaling, the structure on the right ovary appeared to be a granulosa cell tumour; the left ovary was smaller than normal and non-functional. Surgical removal of the right ovary before increasing photoperiod resulted in a return to function of the left ovary and a pregnancy to frozen semen on the second cycle following removal. Figures showing concentrations of inhibin, progesterone, androstenedione, oestradiol and testosterone are presented for this entire period. Unusual ovarian activity in the mare might be a prelude to the development of a granulosa cell tumour.     

Influence of the Preservation Temperature (37, 20, 4, −196°C) and the Mixing of Semen over Sperm Quality of Majorera Bucks

This study assessed the effect of different semen storage temperatures and the influence of semen pooling in semen viability. In experiment 1, semen samples (n = 30) of five Majorera bucks were individually processed [Individual semen (IS)] and after the first dilution (Tris‐yolk extender), semen‐diluted aliquots from each male were pooled semen (PS). Thereafter, semen samples (IS and PS) were preserved as fresh semen (37 and 20°C), chilled semen (4°C) and frozen semen. Sperm motility and the percentage of abnormal sperm cells and intact membrane acrosomes were defined. Semen preservation at 20 and 4°C did not modify the quality of spermatozoa for the first 24 h, but the conservation at 37°C caused a dramatic fall in the semen motility from 12 h onwards. Furthermore, the longevity of frozen‐thawed semen was limited to 4–6 h. No differences were observed in semen parameters when PS was compared with semen from individual males in any of the preservation protocols assessed. In experiment 2, 120 goats were distributed in four experimental groups: in group fresh individual semen (FIS, n = 30) and group frozen‐thawed individual semen (FTIS, n = 30), does were transcervically inseminated with fresh semen and frozen‐thawed semen from each individual male, respectively, and in group fresh pooled semen (FPS, n = 30) and group frozen‐thawed pooled semen (FTPS, n = 30), goats were transcervically inseminated with FPS and FTPS, respectively. The kidding rate was very close in the FIS and FPS groups (70.0% and 73.7%, respectively), and no significant differences were observed in the fertility rate between FTIS and FTPS. The results of this study confirmed that semen samples may be preserved satisfactorily for 24 h both at 20 and 4°C. In addition, the mixture of semen of different bucks did not significantly modify the semen parameters when compared with semen from individual males.     

Follicle culture enhances fertilizability and cleavage of bovine oocytes matured in vitro

The fertilization and cleavage of bovine oocytes matured by intra- or extra-follicular methods were investigated. Oocytes were fertilized in vitro or in the rabbit oviduct and cleavage was assessed after in vitro culture of in vitro fertilized oocytes and after in vivo culture (rabbit oviducts) of xenogenously fertilized oocytes. The effect of fertilization with fresh-diluted or frozen-thawed semen were also examined. The intra-follicular method did not increase the nuclear maturation rate as compared with the extra-follicular method (57.9 and 52.7%, respectively). However, the proportions of in vitro fertilized eggs (54.8%) and of cleaved eggs (two to eight cells; 34.6%) in the rabbit oviduct for 48 h after xenogenous fertilization were higher (P less than .025) in the intra-follicular oocytes than those of the extra-follicular oocytes (37.1 and 21.3%, respectively). It was also found that the use of fresh-diluted semen resulted in more cleaved eggs from the rabbit oviduct than the use of frozen-thawed semen (43.4 and 23.3% in the intra-follicular oocytes, P less than .025; 31.0 and 7.8% in the extra-follicular oocytes, P less than .05), while the appearance of cleaved eggs following in vitro fertilization was extremely low (0 to 6.6%). The present results demonstrated that the intra-follicular culture method of bovine oocytes provided a physiological environment for cytoplasmic maturation leading to higher fertilizability and development than the conventional in vitro culture of extra-follicular oocytes.     

Methods of Concentrating Stallion Semen

Removal of excess seminal plasma is sometimes necessary to increase the quality and the longevity of cooled equine semen; moreover, this procedure is an indispensable step aiming to concentrate the sperm cells before freezing equine semen. Typically, the removal of seminal plasma is achieved by centrifugation; however, studies have shown that the force and duration of centrifugation can damage sperm cells and reduce the sperm recovery rate. Recently, new methodologies, such as cushion and filtration, have been described that aim to decrease the mechanical damage of centrifugation to sperm cells. This study aims to compare different methods for concentrating stallion semen.     

Intracellular calcium chelating agent (BAPTA‐AM) aids stallion semen cooling and freezing–thawing

This study aimed to investigate the effects of different concentrations of 1,2‐bis‐(o‐aminophenoxy)‐ethane‐N,N,N0 N0‐tetraacetic acid, tetra‐acetoxymethyl ester (BAPTA‐AM), an intracellular calcium chelating agent, on stallion semen cooling and freezing–thawing. After collection, semen was extended (1:1 v/v) on a skim milk‐based extender, centrifuged and resuspended at 400 million/ml into cooling or freezing extenders containing 0, 5, 25, 50, 100 and 200 μΜ BAPTA‐AM. Motility parameters were assessed after cooling in Equitainer at 5°C for 12, 24, 48, 72 and 120 hr and after freezing–thawing. In addition, mitochondrial membrane potential, intracellular ATP, reactive oxygen species and malondialdehyde concentrations were measured in cryopreserved‐thawed semen. Cooled stored (48 hr) semen containing 50 μΜ BAPTA‐AM and control extender (0 μΜ BAPTA‐AM) was used to assess fertility. Inclusion of 50 μΜ BAPTA‐AM resulted in superior sperm motility parameters during cooled storage when compared to other groups (p < 0.05). Furthermore, semen cryopreserved in extender containing 50 μΜ BAPTA‐AM showed increased intracellular ATP and mitochondrial membrane potential, whereas reactive oxygen species and malondialdehyde were increased after thawing for all groups (p < 0.05). Addition of 50 μΜ BAPTA‐AM to cooling extender resulted in similar pregnancy rates to the control group (75% vs. 73.6%, respectively; p > 0.05). In conclusion, the addition of BAPTA‐AM to semen extenders aided stallion semen cryopreservation in a dose‐dependent manner. Furthermore, the cooling extender supplemented with 50 μΜ BAPTA‐AM could be used to prolong the sperm motility during cooling without apparently compromising fertility. Field trials should be conducted to assess fertility of cryopreserved stallion semen with BAPTA‐AM.     

Successful use of deep-frozen stallion sperm after 23 years of storage at -196 degrees C

A reduction in the motility of the spermatozoa in stallion semen stored in pellet form for 23 years in liquid nitrogen at -196 degrees C could not be seen after thawing. The insemination of a mare with this semen resulted in a normal pregnancy. A normally developed, healthy male foal was born after a gestational period of 321 days.     

Addition of oxytocin to semen extender improves both sperm transport to the oviduct and conception rates in pigs following AI

Oxytocin (OXT) contained in boar semen is known to produce uterine contraction; therefore, we hypothesized that the co‐injection of OXT with sperm would improve artificial insemination (AI) using liquid or frozen‐thawed boar sperm. We initially examined whether OXT added to semen extender improved sperm transport to the oviduct. Although the addition of OXT did not affect the fresh or frozen‐thawed sperm motility or acrosomal integrity, it significantly increased the number of sperm in the oviduct at 6 h after AI injection with OXT, as compared with the control (P < 0.05). Moreover, some sperm were observed in the sperm reservoir of the isthmus in the OXT treatment group, whereas few sperm were observed in the control. When OXT was added to the semen extender immediately prior to AI, the conception rates were significantly higher in both fresh semen and frozen‐thawed semen than in the control group (P < 0.05: liquid, 87.5% vs. 70.5%; frozen‐thawed, 89.8% vs. 75.0%). From these results, we concluded that the addition of OXT to the semen extender assisted in sperm transportation from the uterus to the oviduct, which resulted in improved reproductive performance.