Effect of age and physiological status on sperm storage 24 hours after artificial insemination in broiler breeder hens

1. Uterovaginal junction (UVJ) tissues were collected 24 h after artificial insemination (AI) from 85- and 125-week-old broiler breeder hens in three physiological states: laying hard shell eggs (HS), laying shell-less eggs (SL) and non-laying (NL). This was confirmed by egg production records and visual appraisal of the oviduct at the time of necropsy. 2. Three longitudinal sections of each UVJ were evaluated microscopically and sperm host glands (SHG) were scored in 5 categories: glandular morphology evident but not lumen present, basophilic stained epithelium and no spermatoza present, glands that contained one to five spermatozoa; glands that contained 6 to 20 spermatozoa and glands that contained more than 20 spermatozoa. 3. Laying hens (HS and SL) at 85 weeks of age had significantly more sperm host glands (SHG) containing spermatozoa than NL hens. At 125 weeks of age HS and SL hens had significantly more unscorable glands. 4. The only category that showed no difference between age and physiological status group was the empty category. No significant differences were observed for any gland category in 82- and 125-week-old NL hens. 5. A greater proportion of the 85-week-old group of HS and SL hens had more SHG containing spermatozoa and fewer unscorable glands that the 125-week-old birds. The only category that showed any difference within the SL group was the low category.     

Incidence of Unilateral Fertilizations after Low Dose Deep Intrauterine Insemination in Spontaneously Ovulating Sows under Field Conditions

A new procedure for non-surgical deep intrauterine insemination (DUI) in unrestrained sows hormonally induced to ovulate, has been reported. In comparison with standard artificial insemination (AI), with this procedure, the sperm numbers inseminated can be reduced 20-fold without reducing the reproductive performance of these hormonally treated sows. The present study evaluated, using two experiments, the reproductive performance applying 20-fold different sperm numbers per AI dose using DUI or standard AI in spontaneously ovulating sows, under field conditions. In experiment 1, AI was applied to crossbred sows at 12, 24 and 36 h after onset of spontaneous oestrus using one of the following two regimes: (i) DUI (treatment) with 0.15 x 10(9) fresh boar spermatozoa in 5 ml of Beltsville thawing solution (BTS) extender (n = 95), and (ii) standard cervical AI (control) with 2.85 x 10(9) fresh spermatozoa in 95 ml of BTS extender (n = 95). The farrowing rates of the two groups of sows were statistically similar (NS). However, a decrease (p < 0.002) in litter size and the total number of pigs born alive was observed in sows inseminated with the DUI procedure. In experiment 2, 42 post-weaned oestrus sows were inseminated following the same design described for experiment 1 during spontaneous oestrus. On day 6 after onset of oestrus, the proximal segment of the uterine horns of the sows were flushed under surgery to retrieve eventual embryos and evaluate the success of fertilization per cornua (e.g. occurrence of effective uni- vs bilateral sperm transport rendering uni- or bilateral, complete or partial fertilization). Retrieved embryos were assessed for cleavage and number of accessory spermatozoa. Although identical overall pregnancy rates were achieved in both insemination groups, the percentage of sows with partial bilateral fertilization and unilateral fertilization was markedly higher (p < 0.05) in the DUI group (35%) compared with the control (standard AI) group (5%), with a consequent lower (p < 0.001) percentage of viable early embryos after DUI. The number of accessory spermatozoa in the zona pellucida of the embryos was highly variable, but higher (p < 0.001) in control animals than in DUI-AI. No accessory spermatozoa were found in oocytes retrieved from sows depicting unilateral fertilization. In conclusion, DUI in spontaneously ovulating sows with 0.15 x 10(9) spermatozoa renders similar farrowing rates but a lower litter size compared with use of standard AI with a 20-fold higher sperm dose. The lower litter size ought to be related to a decreased distribution of spermatozoa after DUI leading to a higher incidence of partial bilateral and unilateral fertilization.     

Storage of sperm in the uterovaginal junction and its incidence on the numbers of spermatozoa present in the perivitelline layer of hens' eggs

1. The numbers of spermatozoa found in the perivitelline layer (perivitelline spermatozoa) of hens' eggs during a 14-d period after insemination were found to be log-dose dependent (r = 0.99) on the quantities of spermatozoa inseminated intravaginally in these hens (50, 100, 200 or 400 million/female). 2. Highly significant correlations were also observed between the perivitelline spermatozoa and the proportion of uterovaginal sperm-storage tubules containing spermatozoa on day 14 after insemination. 3. These data confirm that the number of perivitelline spermatozoa in eggs laid on day 2 after artificial insemination (AI) are highly correlated with the mean percentages of fertility of its duration over a 14-d or 24-d period. As a consequence, eggs laid by the 10% highest or the 10% lowest females primarily classified on the basis of this variable exhibited on average 99% or 49.7% fertility, respectively, over a two-week period after AI.     

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.     

Hysteroscopic insemination of mares with low numbers of nonsorted or flow sorted spermatozoa

The objectives of this study were 1) to compare pregnancy rates resulting from 2 methods of insemination using low sperm numbers and 2) to compare pregnancy rates resulting from hysteroscopic insemination of 5 x 106 nonsorted and 5 x 106 spermatozoa sorted for X- and Y-chromosome-bearing populations (flow sorted). Semen was collected with an artificial vagina from 2 stallions of known acceptable fertility. Oestrus was synchronised (June to July) in 40 mares, age 3-10 years, by administering 10 ml altrenogest orally for 10 consecutive days, followed by 250 microg cloprostenol i.m. on Day 11. All mares were given 3000 iu hCG i.v. at the time of insemination to induce ovulation. Mares were assigned randomly to 1 of 3 treatment groups: mares in Treatment 1 (n = 10) were inseminated with 5 x 10(6) spermatozoa deposited deep into the uterine horn with the aid of ultrasonography. Mares in Treatment 2 (n = 10) were inseminated with 5 x 10(6) spermatozoa deposited onto the uterotubal junction papilla via hysteroscopic insemination. Mares in Treatment 3 (n = 20) were inseminated using the hysteroscopic technique with 5 x 10(6) flow sorted spermatozoa. Spermatozoa were stained with Hoechst 33342 and sorted into X- and Y-chromosome-bearing populations based on DNA content using an SX MoFlo sperm sorter. Pregnancy was determined ultrasonographically at 16 days postovulation. Hysteroscopic insemination resulted in more pregnancies (5/10 = 50%) than did the ultrasound-guided technique (0/10 = 0%; P<0.05) when nonsorted sperm were inseminated. Pregnancy rates were not significantly lower (P>0.05) when hysteroscopic insemination was used for sorted (5/20 = 25%) and nonsorted spermatozoa (5/10 = 50%). Therefore, hysteroscopic insemination of low numbers of flow sorted stallion spermatozoa resulted in reasonable pregnancy rates.     

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.     

Successful Low Dose Insemination of Flow Cytometrically Sorted Ram Spermatozoa in Sheep

The fertility of ram spermatozoa that had undergone flow cytometric sorting (MoFlo SX) and cryopreservation was assessed after low-dose insemination of synchronized Merino ewes. Oestrus was synchronized with progestagen-impregnated pessaries, PMSG and GnRH treatment. Ewes (n = 360) were inseminated with 1 x 10(6), 5 x 10(6) or 15 x 10(6) motile sorted frozen-thawed (S(1), S(5), or S(15) respectively) or non-sorted frozen-thawed (C(1), C(5) or C(15) respectively) spermatozoa from three rams. An additional group of ewes were inseminated with 50 x 10(6) motile non-sorted frozen-thawed spermatozoa (C(50)) to provide a commercial dose control. The percentage of ewes lambing after insemination was similar for C(50) (24/38, 63.2%), C(15) (37/54, 68.5%), S(15) (38/57, 66.7%), S(5) (37/56, 66.1%) and S(1) (32/52, 61.5%) groups (p > 0.05), but lower for C(5) (19/48, 39.6%) and C(1) (19/55, 34.5%) treatments (p < 0.05). This study demonstrates sorted ram spermatozoa are equally fertile to non-sorted spermatozoa even when inseminated at 2% of the dose. Furthermore, at very low artificial insemination doses (1 or 5 million motile) the fertility of sorted ram spermatozoa is superior to non-sorted spermatozoa inseminated in equal numbers. These results have significance for the future commercialization of sex-preselection technology in sheep as a reduction in the minimum effective sperm number will allow a corresponding decrease in the associated cost per dose.     

Birth of puppies after intrauterine and intratubal insemination with frozen-thawed canine semen

The present study was performed to assess the fertility of frozen-thawed dog semen prepared by freezing with 6% glycerol and thawing at 70℃ for 8 sec, and to evaluate the least number of post-thaw spermatozoa necessary to achieve pregnancy by intrauterine or intratubal artificial insemination. It was found that the pregnancy rate of intrauterine artificial insemination was 100% using 6% glycerol buffer and thawing at 70℃ for 8 sec with 5 × 10⁷ spermatozoa. Even though the pregnancy rate (80%) and the whelping rate (24.5%) in the 5 × 10⁶ spermatozoa inseminated group were lower than those of the 5 × 10⁷ spermatozoa group, conception was confirmed with 5 × 10⁶ spermatozoa. Although the pregnancy rate of intratubal insemination was low (20%) with 4 × 10⁶ spermatozoa, this study is the first report to show the pregnancy rate of intratubal insemination with frozen-thawed ejaculated canine semen. In order to improve the pregnancy rate with intratubal insemination of canine spermatozoa, it is necessary to investigate the optimal insemination site of the uterine tube, the appropriate number of sperm, and the direct effect of buffer on oocytes.     

Sperm distribution in the reproductive tract of sows after intrauterine insemination

The purpose of the present study was to compare the number of spermatozoa obtained from different parts of the oviducts and the uterine horns of sows after intrauterine insemination (IUI) and conventional artificial insemination (AI), 24 h after insemination. Twelve crossbred (Landrace x Yorkshire) multiparous sows were used in the experiment. The sows were examined for standing oestrus using a back pressure test and were examined every 4 h after standing oestrus by real-time B-mode ultrasonography to estimate the time of ovulation. The sows were allocated to two groups, group I sows (n = 6) were inseminated by a conventional AI technique with 3 x 10(9) motile spermatozoa in 100 ml of extended semen, and group II sows (n = 6) were inseminated by an IUI technique using 1 x 10(9) motile spermatozoa in 50 ml of extended semen. A single dose of AI or IUI was given using the same boar, 8-10 h before the expected time of ovulation during the second oestrus after weaning. Twenty four hours after insemination, the sows were ovario-hysterectomized. The oviducts and the uterine horns were removed and divided into seven parts, the cranial, middle and caudal uterine horns, the utero-tubal junction (UTJ), the cranial and caudal isthmus, and the ampulla. All parts of the reproductive tract were flushed and the spermatozoa were counted using a haemocytometer. The results revealed that the spermatozoa were found in both the oviducts and the uterine horns in all animals. The number of flushed spermatozoa in the UTJ of groups I and II, was 142,500 and 131,167 (p > 0.05), and in the caudal isthmus was 1411 and 1280 (p > 0.05), respectively. The proportion of spermatozoa in different parts of the reproductive tract in relation to the total number of spermatozoa within the tract was not significantly different between groups I and II (p > 0.05). It could be concluded that IUI, with a three-time reduction in the number of spermatozoa used resulted in the same number of spermatozoa to be deposited in the sperm reservoir around ovulation time.     

二甲基乙酰氨在鸡精液冷冻中的作用分析

用二甲基乙酰氨（ＤＭＡ）及甘油作为冷冻保护剂，对鸡精液进行冷冻保存，解冻后观察精子活力，并进行输精试验，发现以ＤＭＡ作为冷冻保护剂冷冻的精子解冻后活力及人工输精后的受精率都显著高于以甘油作冷冻保护剂的结果。经过毒性试验、冷冻试验及输精试验，发现ＤＭＡ的确是一种效果较好的冷冻保护剂。它在许多方面比甘油更为理想。本文还叙述了ＤＭＡ的性质及作用机理     

采用深部输精与常规输精研究不同剂量和有效精子数对经产母猪繁殖力的影响

为探讨不同输精剂量与不同有效精子数对经产母猪繁殖力的影响,使用深部输精和常规输精作为主要配种方式。将发情母猪随机分为2组,A组为深部输精组(n=60),B组为常规输精组(n=60)。对A、B组内母猪分别用不同的输精剂量(Ⅰ:30亿/80 mL、Ⅱ:15亿/80 mL、Ⅲ:15亿/40 mL;n=20)进行输精。结果表明:与常规输精相比,深部输精组的受胎率和分娩率分别为93.33%和88.33%,略高于常规输精组的86.67%和83.33%,但差异不显著(P>0.05);但深部输精可显著提高经产母猪的产仔数和产活仔数,分别提高1.07头和1.15头。无论是深部输精还是常规输精,15亿/80 mL的输精量,均可获得与30亿/80 mL输精量一致的产仔效果,这一结果可以使优秀种公猪的利用率提高1倍,降低了最新国标《GB23238-2009》中对外种猪输精剂量的要求,建议扩大实验并推广使用。     

Studies of gel with immobilized semen by intrauterine endoscopy post-artificial insemination

An extended lifespan of spermatozoa following artificial insemination (AI) can make the timing of insemination less critical, as previously demonstrated with immobilized spermatozoa that are gradually released from an alginate gel. The purpose was to examine the in vivo dissolution of SpermVital (SV) alginate gel over time by endoscopy and secondly to assess spermatozoa quality after incubation of the gel. In vivo endoscopy showed SV gel in the uterus 3, 6, 20 and 24 hr after AI, demonstrating the potential release of spermatozoa to the uterus during this period. In utero ex vivo incubation of the semen demonstrated that high motility and viability of sperm cells was sustained following overnight incubation.     

The Effect of Glycerol Concentrations on the Post‐thaw In Vitro Characteristics of Cryopreserved Sex‐sorted Boar Spermatozoa

The objective of this study was to optimize protocols for the cryopreservation of sex‐sorted boar spermatozoa. In the experiment 1, we evaluated the effects of a standard boar sperm cryopreservation procedure (3% final glycerol concentration) on the in vitro characteristics of sex‐sorted sperm frozen at low sperm concentrations (20 × 10⁶ sperm/ml; S20 group). Non‐sorted spermatozoa frozen at 1000 × 10⁶ (C1000 group) and 20 × 10⁶ (C20 group) sperm/ml were used as the freezing control groups. In experiment 2, the effects of different final glycerol concentrations (0.16%, 0.5%, 1.0%, 2.0% and 3.0%) on post‐thaw quality of the S20 and C20 groups were evaluated. In both experiments, the samples were evaluated prior to freezing (5°C) and at 30, 90 and 150 min after thawing. Experiment 1 indicated that freezing sperm at low concentrations decreased (p < 0.05) the total motility (TM) and progressive motility (PM) at 90 and 150 min after thawing regardless of whether the sperm were sorted or not. However, the sperm membrane integrity was not affected at any evaluation step. Inexperiment 2, significant effects on the TM and PM because of increased glycerol concentrations in the S20 and C20 groups were observed only at 90 and 150 min after thawing. The samples frozen in 3% glycerol showed lower (p < 0.05) TM and PM values when compared to those frozen in the presence of 0.5% and 1% glycerol. In both experiments, non‐sorted control samples displayed higher percentages of spermatozoa with damaged DNA than sorted spermatozoa. In conclusion, the optimization of cryopreservation conditions by decreasing the glycerol concentrations can improve post‐thaw motility of sex‐sorted spermatozoa frozen at low concentrations.     

Effects of sex-sorted spermatozoa on the efficiency of in vitro fertilization and ultrastructure of in vitro produced bovine blastocysts

Frozen-thawed sexed semen from six bulls (Holstein) was used for studying their efficiency in an in vitro fertilization (IVF)-programme and to compare their ultrastructure with in vitro produced bovine blastocysts produced with non-sorted sperm. Progressive motility of sorted spermatozoa, their IVF rate, development of produced blastocysts and the ultrastructure of the blastocysts were analysed. The cleavage rates of sexed sperm of bulls (groups S1, S2 and S4) were significantly lower than that of unsorted control sperm (P < 0.01). Blastocyst development at day 7 of the sexed semen groups varied between 3.5% and 28.8% versus 33.6% for non-sexed semen. The individual blastocyst yield with sexed semen of group S5 (28.8%) was similar to the mean blastocyst production of the non-sexed control spermatozoa (C, 33.6%; P > 0.05). The remaining five sexed sperm groups resulted in significantly lower developmental rates of blastocysts on day 7 (S1, 4.9%; S2, 0%; S3, 0%, S4, 3.5%; S6, 25.8%, P < 0.01). Group S2 showed microbiological contamination in 50% (four of eight) and S3 in 100% of the experiments (eight of eight). Progressive motility of sexed sperm was significantly lower than that of unsorted sperm (S1, 48 +/- 12.0%; S2, 41 +/- 11.9%; S3, 39.0 +/- 9.9%; S4, 42 +/- 4.6%; P < 0.01; S5, 72 +/- 7.1% and S6, 64 +/- 9.3; P < 0.05 versus C 82 +/- 4.6%). The percentage of progressive motile spermatozoa showed a good correlation with the developmental capacity of blastocysts (r(2): >0.70), the regression parameter was significant (P < 0.01). Furthermore, with a straw containing 10 x 10(6) sexed spermatozoa significantly lower number oocytes was fertilized than with the same concentration of non-sexed sperm (P < 0.01). Our results demonstrate that the suitability of sperm sorting for in vitro fertilization (IVF) is lower than no sexed sperm. Our ultrastructural studies showed that blastocysts produced with flow-cytometrically sex-sorted spermatozoa possessed deviations in the number and structure of organelles like mitochondria, rough endoplasmic reticulum (ER) and nuclear envelope. These morphological alterations may be responsible for compromised development that observed in embryos produced with sex-sorted spermatozoa. Thus, we conclude that sperm sex sorting can markedly affect the efficiency of an IVF-programme.     

The effect of the sperm count in the insemination dose and storage time on the fertilizing capability of the ejaculate of roosters

Ejaculate of cooks was diluted by the Wishart and Davtjan diluents at a ratio of 1 : 1 and stored at a temperature of 4 degrees C during 2, 24, 48 and 72 hours before insemination of laying hens by the doses of 25, 50, 75, 100 and 150. 10(6) spermatozoa. With both diluents, egg fertility higher than 90 per cent was gained after 48 hours of keeping the ejaculate in sperm doses of 75 and 100 . 10(6) spermatozoa in the case of both diluents.     

Sperm-egg penetration in laying breeder flocks: a technique for the prediction of fertility.

1. An experiment was conducted to evaluate indices of fertility including the sperm penetration (SP) assay as a technique for the prediction of fertility. Forty-eight males consisting of White Leghorn (WL), New Hampshire (NH), Iraqi Brown (IBr) and Iraqi Barred (IBa) (12 males each) and 64 WL hens were divided at random into 4 groups of 4 replicates of 3 males and 4 females each. 2. At the beginning of each week semen was collected from males and pooled by breed of male. Hens in each breeding group were inseminated once weekly, by breeding group, for 4 consecutive weeks with pooled semen from WL, NH, IBr and IBa males (WLxWL, NHxWL, IBrxWL and IBaxWL). 3. The differences in percentage of dead sperm, acrosomal abnormalities, mass motility, individual motility and spermatocrit between the experimental breeds demonstrated the superiority of WL and NH males in all these quantitative characters of the semen. On the other hand, WL hens inseminated with spermatozoa from NH males had significantly more sperm-egg penetration (SP) holes than WL hens inseminated with spermatozoa from other breeds of males. The breed of males used for insemination affected fertility, hatchability and embryonic mortality. 4. The highest fertility and hatchability and lowest embryonic mortality were observed in eggs laid by hens inseminated with spermatozoa from WL and NH males in comparison with hens inseminated with spermatozoa from Iraqi males. 5. There was a strong positive correlation between SP values and fertility for WLxWL, NHxWL, IBrxWL and IBaxWL. The correlation for all breeds combined was also significant. In addition, SP was also positively correlated with hatchability and negatively correlated with embryonic mortality.     

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.     

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.     

Intrauterine and intravaginal insemination with frozen canine semen using an extender consisting of orvus ES paste-supplemented egg yolk tris-fructose citrate

Our previous report indicated that addition of Orvus ES Paste (OEP) to the extender of frozen canine semen protected acrosomes and maintained sperm motility after thawing. In this study, artificial insemination (AI) using the frozen semen was carried out. The frozen semen was prepared using egg yolk Tris-fructose citrate, and the final concentrations of glycerol and OEP were 7% (v/v) and 0.75% (v/v), respectively. AI was performed during the optimal mating period predicted from the peripheral plasma progesterone level. In intrauterine insemination (IUI), the bitches were laparotomized and 1 x 10(8) spermatozoa were infused into one of the uterine horns. In insemination of non-OEP supplemented semen, 3 x 10(8) spermatozoa were inseminated. In intravaginal insemination (IVI), 10-40 x 10(8) spermatozoa were inseminated. Conception was obtained in nine of 10 bitches (90.0%) that underwent IUI. The number of newborns was from 1 to 7 (mean 3.6 +/- 0.9). The mean ratio of the number of puppies to the number of ovulations in the inseminated uterine horn was 71.8%. The number of puppies did not exceed the number of ovulation in the inseminated uterine horn. Conception using non-OEP supplemented frozen semen was unsuccessful in all four bitches. In IVI, conception was not obtained in any of the six bitches that received insemination of 10 x 10(8) or 40 x 10(8) spermatozoa, but two of three bitches that received insemination of 20 x 10(8) spermatozoa were fertilized. It was shown that a high conception rate can be obtained by IUI using OEP-supplemented frozen canine semen. Developmenmt of a non-surgical method of IUI and a method of freezing canine sperm applicable to IVI is necessary.     

Glutathione supplementation to semen extender improves the quality of frozen-thawed canine spermatozoa for transcervical insemination

The present study was conducted to evaluate whether supplementation of semen extender with glutathione (GSH) can maintain the quality of frozen-thawed canine spermatozoa. Eighteen ejaculates were obtained from 5 dogs and placed in extender (20% egg yolk, Tris, citric acid, lactose, raffinose, antibiotics and 6.5% glycerol) containing 0 (control), 2.5, 5, 7.5 or 10 mM GSH. The samples were cooled to 4 C and then frozen in liquid nitrogen vapor. Motility parameters of the sperm were evaluated at 0, 1, 2, 3, 4, 12 and 24 h after thawing. Sperm motility was higher in the 5 mM GSH group than in the control or 2.5 and 10 mM GSH groups; this effect was observed at 1 to 24 h after thawing (P < 0.05). The 5 mM GSH group had a higher sperm viability index at 12 and 24 h after thawing compared with the other groups (P < 0.05). Acrosome integrity, evaluated at 4 h after thawing, was greater in two of the GSH-treated groups (5 and 10 mM) compared with the control. Lipid peroxidation (LP) levels immediately after thawing were lower in the 5 and 10 mM GSH groups compared with the control, while those at 12 h after thawing did not differ significantly. Frozen-thawed semen in the 5 mM GSH group was used for transcervical insemination of 4 bitches, resulting in delivery of 5 puppies from 2 bitches. These results indicate that supplementation of semen extender with 5 mM GSH was effective in improving motility, longevity and acrosomal integrity and inhibiting LP levels in post-thaw canine spermatozoa, without any adverse impacts on full-term development after transcervical insemination.