Comparison of fertility on intrauterine insemination between cryopreserved ejaculated and cauda epididymal sperm in dogs

The fertility was compared between ejaculated and cauda epididymal sperm sensitized with prostatic fluid in dog after freeze-thawing using the fertility of ova from the contralateral ovary after injection (2 × 10(8) sperm) into dog uterus on the unilateral ovariectomized side, on the basis of the presence or absence of conception. No significant difference was observed in sperm quality after freeze-thawing between the two groups and conception rates were equivalent and low. Therefore, to achieve a high fertility by intrauterine insemination of canine frozen-thawed ejaculated and cauda epididymal sperm, intrauterine insemination on both sides is recommended, rather than insemination with a lot of sperm of the uterine horn on one side.     

Number of Spermatozoa in the Crypts of the Sperm Reservoir at About 24 h After a Low‐Dose Intrauterine and Deep Intrauterine Insemination in Sows

The aim of this study was to investigate the number of spermatozoa in the crypts of the utero‐tubal junction (UTJ) and the oviduct of sows approximately 24 h after intrauterine insemination (IUI) and deep intrauterine insemination (DIUI) and compared with that of conventional artificial insemination (AI). Fifteen crossbred Landrace × Yorkshire (LY) multiparous sows were used in the experiment. Transrectal ultrasonography was performed every 4 h to examine the time of ovulation in relation to oestrous behaviour. The sows were inseminated with a single dose of diluted fresh semen by the AI (n = 5), IUI (n = 5) and DIUI (n = 5) at approximately 6–8 h prior to the expected time of ovulation, during the second oestrus after weaning. The sperm dose contained 3000 × 10⁶ spermatozoa in 100 ml for AI, 1,000 × 10⁶ spermatozoa in 50 ml for IUI and 150 × 10⁶ spermatozoa in 5 ml for DIUI. The sows were anaesthetized and ovario‐hysterectomized approximately 24 h after insemination. The oviducts and the proximal part of the uterine horns (1 cm) on each side of the reproductive tracts were collected. The section was divided into four parts, i.e. UTJ, caudal isthmus, cranial isthmus and ampulla. The spermatozoa in the lumen in each part were flushed several times with phosphate buffer solution. After flushing, the UTJ and all parts of the oviducts were immersed in a 10% neutral buffered formalin solution. The UTJ and each part of the oviducts were cut into four equal parts and embedded in a paraffin block. The tissue sections were transversely sectioned to a thickness of 5 μm. Every fifth serial section was mounted and stained with haematoxylin and eosin. The total number of spermatozoa from 32 sections in each parts of the tissue (16 sections from the left side and 16 sections from the right side) was determined under light microscope. The results reveal that most of the spermatozoa in the histological section were located in groups in the epithelial crypts. The means of the total number of spermatozoa in the sperm reservoir (UTJ and caudal isthmus) were 2296, 729 and 22 cells in AI, IUI and DIUI groups, respectively (p < 0.01). The spermatozoa were found on both sides of the sperm reservoir in all sows in the AI and the IUI groups. For the DIUI group, spermatozoa were not found on any side of the sperm reservoir in three out of five sows, found in unilateral side of the sperm reservoir in one sow and found in both sides of the sperm reservoir in one sow. No spermatozoa were found in the cranial isthmus, while only one spermatozoon was found in the ampulla part of a sow in the IUI group. In conclusion, DIUI resulted in a significantly lower number of spermatozoa in the sperm reservoir approximately 24 h after insemination compared with AI and IUI. Spermatozoa could be obtained from both sides of the sperm reservoir after AI and IUI but in one out of five sows inseminated by DIUI.     

A study on the number of recovered spermatozoa in the uterine horns and oviducts of gilts,after fractionated or non-fractionated insemination

The objective of this study was to compare the number of recovered spermatozoa, in different parts of the uterine horn and oviduct in gilts, after insemination with fractionated (experiment) and non-fractionated (control) liquid stored semen. The number of spermatozoa and volume of backflow was also investigated. Twenty three cross-bred gilts were used in the study. They were divided into 2 groups, a control group (non-fractionated liquid stored semen, n=10) which were inseminated with 100 ml of liquid stored semen containing 3,000 million spermatozoa per dose and an experimental group (fractionated liquid stored semen, n=10) which were inseminated with 50 ml of liquid stored semen, with 3,000 million spermatozoa per dose and followed by another 50 ml of semen dilutor (Beltsville Thawing Solution, BTS). Thereafter, backflow semen was collected and measured every 15 min for a period of 1 hr. Three or 12 hr after insemination, 5 gilts from each group had the uterus, the horn of the uterus, the oviducts and the ovaries removed under general anaesthesia. The horn of uterus and the oviducts were seperated by ligation into 6 segments. All 6 segments were flushed with BTS to collect all spermatozoa within the segment. Recovered spermatozoa were counted, using a haemocytometer and the volume recorded. It was seen that the percentage of spermatozoa in the backflow semen in the experimental group was less than in the control group. The difference was not significant in the gilts that were operated on 3 hr after insemination, the mean number of spermatozoa in the uterine horn and the utero-tubular junction (UTJ) was more in the experimental than in the control group, but less in the isthmus and the ampulla of the oviduct. The gilts which were operated on 12 hr after insemination, had relativity more ovulating gilts in the control group than in the experimental group (3 of 4 gilts compare to 3 of 5 gilts). The control group had more spermatozoa in the oviduct than the experimental group, but less in UTJ and in the horn of the uterus. Again the difference was not significant. It can be concluded that fractionated (experimental) or non-fractionated (control) insemination of semen with the same number of spermatozoa provides no significant difference in the number of spermatozoa either in the horn of the uterus, the UTJ or the oviduct of gilts.     

Effect of male accessory gland secretions on sensitivity of porcine sperm acrosomes to cold shock, initiation of motility and loss of cytoplasmic droplets

Hyaluronidase release was used as an index of acrosomal membrane damage during cold shock of epididymal boar sperm and ejaculated sperm from intact and vesiculectomized boars. Sperm were also incubated with seminal plasma from intact and vasectomized boars to examine the contributions of male accessory gland secretions. Acrosomal membranes of epididymal sperm were more resistant to cold shock than those of ejaculated sperm. Only 36% of the hyaluronidase released by ejaculated sperm was released by the epididymal sperm in spite of similar hyaluronidase content of the sperm. Preincubation of epididymal sperm in seminal plasma from both intact and vasectomized boars increased resistance to cold shock by 60 to 80%. Initial dilution of epididymal sperm with seminal plasma, rather than Ringer-fructose buffer, was associated with low progressive motility and with retention of cytoplasmic droplets. In contrast, acrosomal membranes of ejaculated sperm from intact and vesiculectomized boars exhibited similar sensitivity to cold shock, releasing hyaluronidase capable of forming .20 and .19 mumol N-acetylglucosamine from hyaluronic acid/10(8) sperm in 8 min. Moreover, seminal plasma from vasectomized boars had no effect on acrosomal sensitivity to cold shock of ejaculated sperm from vesiculectomized boars.     

Infiltration of local immune cells in the sow reproductive tracts after intra-uterine and deep intra-uterine insemination with a reduced number of spermatozoa is less than conventional artificial insemination

The present study investigated the infiltration of leukocyte subpopulations in the utero-tubal junction (UTJ) and each part of the oviducts at about 24 hr after intra-uterine insemination (IUI) and deep intra-uterine insemination (DIUI) compared to conventional artificial insemination (CAI) in sows. Fifteen crossbred Landrace x Yorkshire multiparous sows were used (CAI, n=5; IUI, n=5; DIUI, n=5). The sperm dose contained 3,000 × 10(6) (100 ml), 1,000 × 10(6) (50 ml) and 150 × 10(6) (5 ml) motile spermatozoa for CAI, IUI and DIUI, respectively. The sows were inseminated with extended fresh semen at 6 to 8 hr prior to the expected time of ovulation. At 25.2 ± 1.6 hr after insemination, the oviducts and the UTJ were collected. The tissue samples of UTJ, caudal isthmus, cranial isthmus and ampulla were transversely cut to a thickness of 5 μm and stained with H&E. The total numbers of lymphocytes, neutrophils, macrophages, eosinophils and plasma cells were determined under light microscope. It was found that the numbers of lymphocytes, eosinophils and macrophages after CAI, IUI and DIUI were not significantly different (P>0.1) in both epithelial and sub-epithelial connective tissue layer of the UTJ, caudal isthmus, cranial isthmus and ampulla. Intra-epithelial neutrophils in the UTJ were higher than cranial isthmus (P<0.05) and ampulla (P<0.05). In the UTJ, the intra-epithelial neutrophil in the CAI group was higher than DIUI group (P<0.01). Plasma cells in sub-epithelial layer of the endosalpinx in the CAI group were higher than DIUI group (P<0.05) and tended to be higher than the IUI group (P=0.08). In conclusion, compared to CAI, IUI and DIUI do not influence the infiltration of lymphocytes, macrophages and eosinophils in the UTJ and the oviduct prior to fertilization. But a lower number of neutrophils in the intra-epithelial layer of the UTJ and plasma cells in the sub-epithelial layers of the oviduct was observed in the DIUI group compared to CAI.     

Assisted reproduction for felid species conservation—Sperm competences at risk

Cryobanking of gametes in combination with artificial insemination is an essential option to support conservation programmes for endangered and threatened species. About two-thirds of the felid species are classified as ‘near threatened’, ‘vulnerable’ or ‘endangered’ ( www.cites.org ), and mostly, epididymal sperm are collected from euthanized or castrated male felids and cryopreserved. However, epididymal compared with ejaculated and cryopreserved compared with fresh sperm have a limited potential to fertilize if vaginal non-surgical insemination is applied in feline species. Missing or highly diluted seminal fluid in epididymal and cryopreserved sperm, as well as a potential interference of extender ingredients with the natural interactive properties of sperm in the female genital tract is discussed as potential drawback which hampers a proper sperm transit and fertilization besides the limited longevity of cryopreserved feline sperm. Individual components in seminal fluid as well as cryoextenders may adversely alter sperm properties and have a different impact on fertility and preservation success. The identification and investigation of beneficial as well as detrimental components is a precondition to deduce options for improving the process of cryopreservation in felids, particularly, if only epididymal sperm are available.     

Preservation of Epididymal Stallion Sperm in Liquid and Frozen States: Effects of Seminal Plasma on Sperm Function and Fertility

Three separate experiments were conducted to improve preservation of stallion epididymal sperm. In the first one, two different cooling extenders (Kenney and Gent) were compared. Sperm viability and motility patterns were assessed in 10 different epididymal sperm samples after 0 hours, 24 hours, 48 hours, 72 hours, and 96 hours of preservation at 4°C. No significant differences were observed in any of the evaluated parameters either between extenders or throughout the storage period. The second set of experiments was designed to determine whether supplementing thawing medium (INRA Freeze) with seminal plasma had any impact on the quality of frozen-thawed epididymal sperm. Ten epididymal frozen-thawed sperm samples coming from separate stallions were used and different functional parameters (sperm membrane integrity and lipid disorder, motility, intracellular Ca²⁺ levels, and intracellular concentrations of peroxides and superoxides) were evaluated after incubation with or without 50% seminal plasma. Supplementing thawing medium with seminal plasma had no impact on sperm function and survival. The third experiment was an in vivo study. Twenty-five mares were inseminated with epididymal frozen-thawed sperm and seminal plasma, and 21 were bred with epididymal frozen-thawed sperm only. Pregnancy rates obtained for mares artificially inseminated with epididymal frozen-thawed sperm and seminal plasma were significantly (P < .05) higher than those observed when seminal plasma was not infused (64% vs. 19%). Taken together, our data indicate that the quality of epididymal stallion sperm can be maintained at 4°C for up to 96 hours. In addition, not only does supplementing frozen-thawed epididymal sperm with seminal plasma have any damaging effect on their quality but it may also improve pregnancy rates after artificial insemination.     

Ultrastructure of the Uterotubal Junction in Preovulatory Pigs

The ultrastructure of the surface epithelia from the uterotubal junction (UTJ), and the adjacent tubal isthmic and endometrial regions, was studied in preovulatory oestrus gilts, either unmated or inseminated 12 h before with fresh boar semen. The simple columnar epithelium of the UTJ consisted of non-ciliated (secretory) and ciliated cells. Secretory vesicles occurred in the secretory cells, especially in inseminated gilts. Lymphocytes, monocytes and macrophages were found dispersed basally among the epithelial cells. Phagocytosis of epithelial cells undergoing apoptosis was seen throughout the UTJ at oestrus, increasing after insemination. Neutrophilic granulocytes were found in the lamina propria of the uterine component of the UTJ, but only occasionally in the epithelium. After insemination, neutrophils invaded the uterine epithelium, to actively participate in intraepithelial phagocytosis or move into the lumen, engulfing spermatozoa. Neutrophils were absent from the UTJ proper and the isthmic epithelium, irrespective of the presence of spermatozoa in the lumen. Those spermatozoa in the uterine lumen that escaped phagocytosis had severely damaged plasma membranes, whereas those in the UTJ proper--concentrated towards the deep furrows of the diverticulae--mostly showed normal sperm ultrastructure.     

Expression of Progesterone Receptor in the Utero‐tubal Junction After Intra‐uterine and Deep Intra‐uterine Insemination in Sows

The aim of this study was to investigate the expression of progesterone receptor (PR) in the utero‐tubal junction (UTJ) of sows at 24 h after intra‐uterine insemination (IUI) and deep intra‐uterine insemination (DIUI) compared with conventional artificial insemination (AI) in pigs. Fifteen multiparous sows were used: AI (n = 5), IUI (n = 5) and DIUI (n = 5). The sows were inseminated with a single dose of diluted semen during the second oestrus after weaning at 6–8 h prior to ovulation (AI: 3000 × 10⁶ spermatozoa, IUI: 1000 × 10⁶ spermatozoa and DIUI: 150 × 10⁶ spermatozoa). The UTJ was collected and subject to immunohistochemical staining using avidin‐biotin immunoperoxidase technique with mouse monoclonal antibody to PR. In the oviductal part of the UTJ, the intensity of PR in the tunica muscularis and the proportion of PR‐positive cells in the surface epithelium after DIUI were lower than AI (p < 0.05). The intensity and the proportion of PR‐positive cells between AI and IUI in all compartments of the UTJ did not differ significantly (p > 0.05). When comparing between tissue compartments, prominent staining was observed in the muscular layer of the UTJ. It could be concluded that the expression of PR in the UTJ prior to fertilization after DIUI with a reduced number of spermatozoa was lower than that after AI. This might influence sperm transportation and the fertilization process.     

Tubale Befruchtung von Kaninchen-Eizellen mit Nebenhodensperma

Abstract: Fertilization after tubal insemination with epididymal sperm in the rabbit
Sperm from the distal corpus and proximal cauda epididymides was introduced into the ligated oviduct of females treated with HCG to induce ovulation. Of the ova recovered 50% (distal caput) and 94% (proximal cauda) respectively, were fertilized.     

Artificial insemination of frozen epididymal sperm in beagle dogs

Freeze-storage of epididymal sperm is an important technique for the preservation of gametes in animals, including those becoming extinct. We froze canine sperm recovered from the cauda epididymis and investigated the fertility. The qualities of sperm from the cauda epididymis before freezing were: mean sperm motility, 89.4 +/- 1.6 (SE) %; sperm viability, 89.1 +/- 1.1%; and these were significantly higher than those of sperm from the caput-corpus epididymis (P<0.01, P<0.05). The number of sperm recovered from both cauda epididymides varied among animals: 6.3-122.3 x 10(7), mean 61.5 +/- 10.0 x 10(7). Freezing was used only for sperm recovered from the cauda epididymis. The sperm motility and viability after thawing were 19.5 +/- 2.5% and 53.1 +/- 3.3%, respectively. These were slightly lower than those of frozen-thawed ejaculated sperm, but the differences were not significant. When 2 x 10(8), 3 x 10(8), or 4 x 10 (8) sperm were inseminated in the unilateral uterus, only one animal inseminated with 3 x 10(8) sperm was fertilized (1/16, 6.3%). When 1 x 10(8) sperm were inseminated in the bilateral uterine tubes, one of six animals (16.7%) was fertilized. Therefore, although the qualities of epididymal sperm after thawing were similar to those of ejaculated sperm, the conception rate obtained with frozen-thawed epididymal sperm was low in beagle dogs. It is necessary to investigate the differences in damage between epididymal sperm after thawing and ejaculated sperm and to develop a method for improving the conception rate.     

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.     

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.     

Influence of Sperm Cell Dose and Post-insemination Backflow on Reproductive Performance of Intrauterine Inseminated Sows

This study was carried out to evaluate the effects of the sperm cell dose and semen backflow on the pregnancy rate and number of embryos of sows inseminated once at 0-24 h before ovulation, using an intrauterine technique. The results were analysed from a total of 211 sows assigned to three groups inseminated with doses of 0.25 x 10(9) (T1), 0.5 x 10(9) (T2) and 1.0 x 10(9) (T3) spermatozoa. Semen backflow was observed in 95% of the females (143/151) evaluated for this purpose. The percentage of semen backflow is close to two-third of the volume and the percentage of sperm is around 15% of the infused sperm dose. Intrauterine insemination can be successfully performed provided that at least 0.5 billion of sperm cell dose is infused at an interval of 0-24 h before ovulation.     

Porcine Field Fertility with Two Different Insemination Doses and the Effect of Sperm Morphology

In swine artificial insemination, several dose regimens are applied, ranging from 1.5 x 10(9) to 6.0 x 10(9) spermatozoa per intra-cervical insemination dose. A lower sperm dose is more profitable for artificial insemination centres and offers a more effective use of superior boars. To evaluate fertility, 50 boars were used for a total of 10 773 homospermic first inseminations at a dose of 2 billion spermatozoa. In addition, 96 boars were used at a dose of 3 billion spermatozoa for 34 789 homospermic first inseminations. Fertility was determined by a 60-day non-return rate (NR%) of first inseminations. Litter size was registered by total number of piglets born separately in primiparous and multiparous farrowings. On average, a sow was inseminated 1.5 times. A significant decrease was observed in all three fertility parameters (NR%, litter size of both primiparous and multiparous farrowings) with a dose of 2 billion spermatozoa compared with a dose of 3 billion spermatozoa. The NR% was 75.8% and 84.0% (p < 0.001), the mean litter size of primiparous farrowings 10.1 and 10.7 (p < 0.001) and the mean litter size of multiparous farrowings 11.7 and 12.1 (p < 0.001) for 2 and 3 billion spermatozoa/dose, respectively. The proportion of normal spermatozoa in the sperm morphology analysis correlated significantly with NR% in both insemination regimens: p < 0.001, r = 0.604 and p < 0.05, r = 0.223 for 2 and 3 billion spermatozoa/dose, respectively. These results confirm that quantity can at least partly compensate for poor sperm quality. When the boars with <70% normal spermatozoa in the morphology evaluation were excluded from the data there were no correlation between the sperm morphology and NR%. However, the difference between the NR% and litter size remained statistically significant (p < 0.001) in favour for the bigger insemination dose. In conclusion, a decrease in sperm dose from 3 to 2 billion spermatozoa on commercial farms will severely decrease prolificacy at least under field conditions, where a sow is inseminated an average of 1.5 times/heat, and the semen is typically used within 3 days after collection. We recommend that under commercial circumstances the homospermic semen doses contain no <3 billion spermatozoa/dose.     

Is the Function of the Porcine Sperm Reservoir Restricted to the Ovulatory Period?

The uterotubal junction (UTJ) and caudal isthmus are recognized as a functional pre-ovulatory sperm reservoir (SR). Spermatozoa are released from the SR in a complex and concerted action. However, whether this functionality is restricted only to the ovulatory period is still open to debate. Our study was aimed to analyze the presence of spermatozoa within the UTJ (SR), isthmus (ISTH) and ampulla (AMP) after laparoscopic intrauterine insemination (LIUI) either in the peri- (PERI) or post-ovulatory (POST) period or at mid cycle (MID). Each uterine horn of estrus synchronized gilts (n=12) was inseminated with 20 ml sperm (29.5×10⁶ cells/ml). Oviducts were recovered 7 h after LIUI and separated into the UTJ, ISTH and AMP, and sections were flushed with 10 ml PBS+EDTA solution. After centrifugation, the sperm pellet was evaluated by Čeřovský staining. The median sperm numbers in the PERI, POST and MID groups were 578, 171 and 789 in the UTJ; 545, 233 and 713 in the ISTH; and 496, 280 and 926 in the AMP, respectively, and there were differences between the POST and MID groups (P<0.05) but not between the oviductal sections of each group (P>0.05). Compared with the MID group, the percent of intact sperm cells was higher (P<0.01) in the PERI and POST groups (32.8 vs. 66.4 and 76.8%). Also, the percentages of aberrations in the acrosome and tail were higher (P<0.05) in the MID group. Based on this, it can be assumed that the sperm reservoir is active during different phases of the estrus cycle. However, the mid-cycle oviduct environment considerably impairs sperm cell quality.     

The chemotactic properties of porcine seminal components toward neutrophils in vitro

Our objectives were to investigate the mechanisms of postbreeding inflammation in swine by examining the chemotactic properties of polymorphonuclear neutrophilic granulocytes (PMN) and of various populations of spermatozoa and seminal plasma. Epididymal spermatozoa from two boars obtained under sterile conditions, washed ejaculated spermatozoa from two boars, and pooled seminal plasma from eight boars of known fertility were examined for chemotaxis to PMN. The chemotaxis of blood-derived PMN in response to sperm and seminal plasma was evaluated and expressed as a percentage of a positive control (lipopolysaccharide-activated blood plasma). The mean chemotactic effect of washed sperm alone (4.4+/-0.04) and of epididymal sperm alone (3.4+/-0.06) was not different from that of the negative controls (3.1+/-0.05) of McCoy's medium with 10% heat-inactivated fetal calf serum. A marked chemotactic effect was detected when washed ejaculated and epididymal sperm were incubated with blood plasma, compared with blood plasma without spermatozoa (P < 0.001). Washed sperm in blood plasma (86.2+/-5.6) and epididymal sperm in blood plasma (83.9+/-7.7) were different from blood plasma alone (11.2+/-1.5), but no differences were detected between the two populations of sperm. This effect, however, was not completely inhibited by heat inactivation of the blood plasma. The chemotactic response of washed ejaculated and epididymal spermatozoa incubated in lipopolysaccharide-treated, heat-inactivated blood plasma were greater than that of the negative control (P < 0.05). Polymorphonuclear neutrophilic granulocyte migration toward seminal plasma was similar to the negative control (4.0+/-0.04 vs 3.1+/-0.05). It seems that porcine epididymal sperm and ejaculated sperm activate chemotactic components in porcine blood plasma and heat-inactivated blood plasma, suggesting that, at least partially, a heat-stable (noncomplement) blood plasma component may be involved in sperm-induced PMN chemotaxis. In contrast, porcine seminal plasma was not chemotactic to PMN. These results support the hypothesis that spermatozoa play an active role in initiating postbreeding endometritis.     

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.     

Production of Diabetic Offspring Using Cryopreserved Epididymal Sperm by In Vitro Fertilization and Intrafallopian Insemination Techniques in Transgenic Pigs

Somatic cell nuclear transfer (SCNT) is a useful technique for creating pig strains that model human diseases. However, production of numerous cloned disease model pigs by SCNT for large-scale experiments is impractical due to its complexity and inefficiency. In the present study, we aimed to establish an efficient procedure for proliferating the diabetes model pig carrying the mutant human hepatocyte nuclear factor-1α gene. A founder diabetes transgenic cloned pig was generated by SCNT and treated with insulin to allow for normal growth to maturity, at which point epididymal sperm could be collected for cryopreservation. In vitro fertilization and intrafallopian insemination using the cryopreserved epididymal sperm resulted in diabetes model transgenic offspring. These results suggest that artificial reproductive technology using cryopreserved epididymal sperm could be a practical option for proliferation of genetically modified disease model pigs.     

Artificial insemination with frozen epididymal sperm in cats

Artificial insemination with frozen cauda epididymal sperm was performed in cats. Sperm were transmigrated from the epididymides in 10 male cats. The mean sperm motility and viability were 67% and 82.5%, respectively, and 11.6 x 10(7) sperm were recovered. The mean sperm motility after thawing was 24.0%. Eleven female cats received unilateral intrauterine insemination of 5 x 10(7) sperm, and the conception rate was 27.3% (3/11). This was the first case of conception obtained with frozen epididymal sperm in cats.