Influence of Inseminate Components on Porcine Leucocyte Migration In Vitro and In Vivo after Pre- and Post-Ovulatory Insemination

A post‐breeding migration of leucocytes (PMN) into the uterus is considered to be an important reason for sperm losses. Minimizing such effects may be necessary for successful insemination with low sperm numbers, as required with sex‐sorted spermatozoa. We examined the magnitude of PMN influx 3 h after pre‐ or post‐ovulatory insemination with various combinations of seminal plasma (SP), semen extender Androhep? (AH; Minitüb, Tiefenbach, Germany) and sperm preparations (S). Pre‐ovulatory inseminations with preparations containing 98% AH caused a massive influx of PMN, independent of whether spermatozoa were present (628 ± 189 × 10⁶ leucocytes/uterine horn) or not (580 ± 153 × 10⁶). Post‐ovulatory, 98% AH caused a comparable immigration only in the absence of sperm cells (AH: 569 ± 198 × 10⁶, AH+S: 162 ± 102 × 10⁶). The presence of SP significantly dampened the numbers of recruited uterine leucocytes. The reaction to all inseminates containing 98% SP both with and without spermatozoa, used before ovulation (SP: 14 ± 6 × 10⁶, SP+S: 73 ± 27 × 10⁶) and after ovulation (SP: 60 ± 32 × 10⁶, SP+S: 51 ± 33 × 10⁶) did not differ significantly from controls using phosphate buffered saline (PBS) (pre‐ovulatory: 1 ± 1 × 10⁶, post‐ovulatory: 11 ± 9 × 10⁶). Quantitative in vitro transmigration assays with blood‐derived PMN proved that AH‐induced leucocyte migration into the uterus to be not as a result of direct chemotaxis, because, on account of the chelator citrate, AH significantly inhibited the transmigration towards recombinant human Interleukin‐8 (rhCXCL8) (AH: 14 ± 5% migration rate vs controls: 37 ± 6%, p < 0.05). Supernatants of spermatozoa incubated in PBS for 1, 12 or 24 h showed neither chemoattractive nor chemotaxis‐inhibiting properties. SP at ≥0.1% [v/v] significantly inhibited the in vitro transmigration of PMN. With respect to in vivo migration of neutrophils, the striking difference in the results between semen extender and seminal plasma suggests that adaptation of extender composition is needed to reflect more closely the in vivo regulatory potential of natural seminal plasma.     

Interaction of Intact Porcine Spermatozoa with Epithelial Cells and Neutrophilic Granulocytes during Uterine Passage

New insemination techniques allow a tremendous sperm reduction for successful artificial insemination (AI) if highly diluted semen is deposited in the tip of the uterine horn and close to the utero‐tubal junction. High sperm losses are known to occur during uterine passage and it was the general question whether specific binding mechanisms are involved. Upon arrival in the uterus, spermatozoa are confronted with mainly two different cell types: uterine epithelial cells (UEC) and neutrophilic granulocytes (polymorphonuclear neutrophil, PMN). As cell–sperm interactions can hardly be observed in vivo, an ex vivo system was established to study the interaction between spermatozoa and the UEC. Uterine segments (10 cm) from freshly slaughtered synchronized juvenile gilts were inseminated for 60 min at 38°C. Thereafter spermatozoa were recovered, counted flow cytometrically and examined for changes in viability and mitochondrial membrane potential (MMP). Significantly less spermatozoa with a functioning MMP and intact plasma membranes could be retrieved (55 ± 7%), while the number of damaged spermatozoa hardly changed (93 ± 12%), indicating retention of viable sperm cells in the uterine lumen. The interactions between porcine PMN and spermatozoa (motile, immotile, membrane‐damaged) were studied in coincubation assays in vitro. The binding of membrane‐damaged sperm cells to PMN was virtually non‐existent (3 ± 2%). Viable and motile spermatozoa attached to PMN without being phagocytosed within 60 min (45 ± 3%), whereas binding to sodium fluoride (NaF)‐immobilized spermatozoa was reduced to 20 ± 2%. The binding of viable sperm to PMN is most likely not lectin‐dependent; although both viable cell types were shown to express a broad range of different lectin‐binding sugar residues, none of the lectins tested was able to selectively block PMN‐sperm binding significantly. The results of the study suggest that viable spermatozoa are already subject to selective processes within the uterus before further selection is initiated at the utero‐tubal junction and in the oviductal isthmus.     

Effect of seminal plasma on uterine inflammation, contractility and pregnancy rates in mares

REASONS FOR PERFORMING STUDY: There is conflicting evidence over the role seminal plasma plays in sperm transport and inflammation within the uterus of mares. In in vitro studies, seminal plasma has been shown to reduce polymorphonuclear neutrophil (PMN) function, but the opposite effect on uterine inflammation has been reported in vivo. OBJECTIVES: To study the effect of seminal plasma on uterine contractility, inflammation and pregnancy rates by inseminating mares with low doses of sperm free from seminal plasma (Group 1) and containing seminal plasma (Group 2). METHODS: Synchronised mares were inseminated with 50 x 10(6) sperm in either skim milk extender or seminal plasma. Uterine lavage was performed 6 h after insemination to assess the inflammatory response. The contraction frequency of the uterus was measured over a 4 min period 10 mins and 6 h after insemination, using B-mode ultrasonography. Pregnancy rates were assessed 16 days after insemination. RESULTS: Uterine contractions were less frequent in Group 1 mares inseminated with seminal plasma and significantly more PMNs were found in the lavage fluid of those mares. Pregnancy rates were identical in both groups (62%). CONCLUSIONS: This study provides evidence that seminal plasma decreases uterine contractility and increases the inflammatory response of the uterus to semen. No effect of seminal plasma on pregnancy rates was demonstrated. POTENTIAL RELEVANCE: Mares that develop persistent mating-induced endometritis may have inherently poor uterine contractility and impaired uterine clearance. The presence of seminal plasma during breeding may not be desirable in these mares. The role of seminal plasma in problem mares warrants additional study.     

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.     

The effect of spermatozoa and seminal plasma on leukocyte migration into the uterus of gilts.

Yorkshire x Landrace gilts were used to determine the effect of spermatozoa and seminal plasma on postbreeding uterine leukocyte influx. Estrus detection was performed with a boar at 12-h intervals following synchronization with 400 IU eCG and 200 IU of hCG. All gilts were AI once, 24 h after the detection of estrus following random assignment to a 2x2x3 factorial arrangement of treatments (sperm or sperm-free AI doses), AI dose medium (seminal plasma or PBS), and lavage time following AI. Gilts were treated with sperm (5x10(9) spermatozoa; SPZ; n = 30) or sperm-free (SF; n = 30) doses containing either 100 mL of seminal plasma (SP; n = 15/treatment) or PBS (n = 15/treatment). Uterine lavage was performed once on each gilt (n = 20/time) at one of three times after AI (6, 12, or 36 h) to determine the total number of uterine leukocytes. The leukocytes consisted predominately (92 to 99%) of polymorphonuclear neutrophilic granulocytes (PMN). There was an AI x medium interaction on uterine PMN numbers. The number of uterine PMN recovered from gilts inseminated with sperm suspended in PBS was greater than the number of PMN recovered from the uterine lumen of gilts inseminated with sperm in SP, SP alone, or PBS alone (P<.05). Furthermore, SP accelerated the rate of uterine clearance when suspended with sperm cells during the first 36 h following AI (P<.05). These results indicate that seminal plasma suppresses PMN migration into the uterus following breeding and enhances the rate of disappearance of uterine inflammation.     

Distribution of Live and Dead Spermatozoa in the Genital Tract of Gilts at Different Times After Insemination

Twenty-four gilts were inseminated pair-wise with live or dead spermatozoa from the same ejaculate. The insemination dose was 100 ml undiluted semen containing, on average, 19×10⁹ spermatozoa. The gilts were slaughtered 1, 2, 6 and 12 h after insemination. The numbers of spermatozoa were counted in the uterus, uterotubal junction and in four equally long segments of the oviduct, called I–IV, with a haemocytometer. IV was adjacent to the uterotubal junction. The numbers of spermatozoa recovered in the uterus diminished significantly during the first 12 h after insemination. From gilts inseminated with live spermatozoa more spermatozoa were recovered in the uterotubal junction than from gilts inseminated with dead spermatozoa. Two h after insemination spermatozoa were recovered in all oviducts. Significantly more live than dead spermatozoa were recovered in Segments III and IV of the oviduct, regardless of time. In gilts inseminated with live spermatozoa the sperm count in Segment I varied significantly with time, being hiigest 2 h after insemination. At 6 and 12 h there were no distinct differences in the distribution of live spermatozoa between the various oviduct segments. The numbers of spermatozoa recovered in the oviduct were at these times apparently related to the sperm depots in the uterotubal junction.     

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.     

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.     

Effects of Seminal Fluid Fractions on Plasma and Acrosome Membrane Integrity and Mitochondrial Membrane Potential Determined by Flow Cytometry in Chilled Canine Spermatozoa

Depending on the mammal species, the use of seminal plasma during semen processing for cryopreservation has been found to have both beneficial and detrimental effects. This study was designed to determine the effects of the second (SF) and third [prostatic fluid, (PF)] ejaculate fractions on plasma membrane and acrosome integrity, mitochondrial membrane potential (ΔΨm), phosphatidylserine (PS) translocation and sperm motility in chilled canine spermatozoa by flow cytometry. After pooling the second sperm‐rich fraction of ejaculates from six dogs, samples for each assay were preserved at 5°C for 72 h in egg yolk‐TRIS extender (EYT) alone (control) or supplemented with seminal fluid from the second (EYT‐SF) or third (EYT‐PF) ejaculated fractions. After cold storage, groups EYT‐SF and EYT‐PF showed significantly higher percentages of sperm cells with an intact acrosome [68.8 ± 1.4%, 69.6 ± 2.6% (p < 0.01)] and intact plasma membrane [48.1 ± 2.8%, 50.4 ± 8.2% (p < 0.001)] than that observed in EYT [51.7 ± 3.2% and 33.3 ± 4.1% respectively]. Only in EYT‐SF was PS translocation significantly reduced compared to EYT‐PF and EYT [3.9 ± 0.4%, 10.2 ± 2.2% and 9.0 ± 1.5%, respectively (p < 0.001)]. However, significantly diminished sperm motility was observed in EYT‐SF and EYT‐PF compared to EYT [36.8 ± 2.1%, 35.5 ± 2.3% and 78.4 ± 4.7% (p < 0.001)]. No significant differences were detected in ΔΨm (p > 0.05). In conclusion, supplementing semen extenders with seminal fluid from the second or third fractions of the ejaculate supplementation helps to preserve the integrity of the plasma and acrosome membranes along with the mitochondrial membrane potential but seems to compromise the motility of canine spermatozoa chilled for 72 h.     

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.     

肝素调控猪嗜中性粒细胞对精子的趋化和吞噬作用

为降低由人工授精诱导的猪子宫局部免疫反应,从而提高其人工授精效率,在含有10%血清的培养液中添加不同浓度的肝素(0、100、500、1000μg/mL),检测猪嗜中性粒细胞(PMNs)对猪精子的吞噬性和趋化性。结果表明,100~1000μg/mL的肝素可显著抑制猪PMNs对精子的吞噬性(P<0.01)和趋化性(P<0.05)。提示肝素可作为猪精液稀释液的保护成分,降低由人工授精所导致的子宫炎症反应,从而提高猪的人工授精受胎率。     

Effect of collection frequency on quantitative and qualitative characteristics of pigeon (Columba livia) semen

The aim was to estimate the optimal frequency of semen collection from pigeons in relation to ejaculate volume, sperm concentration, total spermatozoa in ejaculate and percentage of live morphologically normal cells. The study was carried out on 455 ejaculates collected from two groups of pigeons, each of 10 males (group I: meat-type breed; group II: fancy pigeon). The birds were selected and kept individually in cages under a natural photoperiod. A two-person technique was used for semen collection (lumbo-sacral and cloacal region massage). Semen was collected once, twice or three times per week. Colour, consistency and volume of ejaculates were evaluated macroscopically immediately after collection. Sperm concentration and total number of cells in the ejaculate were estimated after dilution with Ringer's solution. A live-dead stain technique (nigrosin-eosin) was used to determine the percentage of live and normal spermatozoa. Semen collected 3x/week was of high quality. The average volume of a single ejaculate was small (21 microl in group I and 19 microl in group II), but sperm concentration was high--1.58 x 10(9)/ml and 1.96 x 10(9)/ml, respectively. The mean number of spermatozoa per ejaculate was 30.48 x 10(6) in group I and 39.49 x 10(6) in group II. An increased percentage of live and normal spermatozoa in semen collected more frequently was also observed. Collecting pigeon semen 3x/week provides spermatozoa in larger amounts and of better quality than less frequent collections (1x/week or 2x/week) and is recommended for obtaining more insemination doses.     

Sperm Production and Sperm Morphology of Swedish Warmblood Stallions

The present study investigated daily sperm output and sperm morphology of fresh semen in eight Swedish Warmblood stallions aged 5–8 years. They were used for artificial insemination, and their fertility during the breeding season of semen collection exceeded 60% per cycle. One ejaculate of semen was collected daily for 10 consecutive days from each stallion. The gel-free volume was measured, and the sperm concentration was assessed with a Bürker chamber. The volume of gel-free fraction was multiplied by the sperm concentration to give the total number of spermatozoa (TSN). Sperm morphology was examined in ejaculates collected on days 2, 5 and 10. An aliquot from each ejaculate was fixed in 1 ml formol–saline immediately after collection and examined under a phase-contrast microscope (magnification 1000×) to assess morphological abnormalities. Furthermore smears were prepared and stained according to Williams (carbolfuchsin–eosin) for a more detailed examination of the sperm heads under a light microscope (magnification 1000×). Analysis of variance was applied to data. Total spermatozoa number decreased progressively during the first 8 days of collection, and daily sperm output (DSO) was calculated as mean TSN of collections on days 8–10, being 6.4 × 10⁹ spermatozoa. The overall percentages of morphologically normal spermatozoa in ejaculates collected on days 2, 5 and 10 were above 70%, being significantly lower in ejaculate 2 (68.6%) compared with ejaculates 5 and 10 (72.9% respectively 75.3%).     

Effects of coitus and the artificial insemination of different volumes of fresh semen on uterine contractions in mares

Uterine contractions may play an important role in the transportation of spermatozoa towards the site of fertilisation in the oviduct of mares. M-mode ultrasound was used to measure the number, amplitude and duration of uterine contractions in each uterine horn and the uterine body of oestrous mares for four minutes before and four minutes after either coitus, or the artificial insemination of either 80.0 ml of fresh semen or 10.0 ml of fresh semen. The direction of the uterine contractions in each uterine horn and the uterine body was measured before and after coitus. Coitus and the insemination of 80.0 ml semen significantly increased the total number, mean amplitude and mean duration of contractions in all parts of the uterus. The insemination of 10.0 ml of semen did not affect the total number or the mean duration of contractions in the uterine horns. Their mean amplitude was increased, but largely owing to the results from one mare; it also did not affect the contractions in the uterine body. There was no significant difference between the percentage of contractions moving in a cervicotubal or tubocervical direction after coitus in any part of the uterus examined.     

Combined cryopreservation of canine ejaculates collected at a one-hour interval increases semen doses for artificial insemination without negative effects on post-thaw sperm characteristics

A limiting factor in canine artificial insemination (AI) is the low number of insemination doses obtained per ejaculate. In this study, semen was collected from dogs (n = 28) either once and frozen directly after collection or the same dogs were submitted to a dual semen collection with a 1-hr interval and the two ejaculates were combined for cryopreservation. We hypothesized that combining two ejaculates increases semen doses per cryopreservation process without negative effects on semen characteristics. Total sperm count was lower in semen from a single semen collection in comparison with the combination of the first and second ejaculate of a dual semen collection (p < .001). The percentage of motile and membrane-intact spermatozoa determined by computer-assisted sperm analysis (CASA) in raw semen did not differ between single and combined dual ejaculates and was reduced (p < .001) by cryopreservation to the same extent in single (motility 73.7 ± 1.8%, membrane integrity 65.6 ± 2.2%) and combined dual ejaculates (motility 72.7 ± 2.3%, membrane integrity 64.6 ± 2.5%). The percentage of spermatozoa with morphological defects increased after cryopreservation (p < .001) but was similar in single and combined dual ejaculates. The CASA sperm velocity parameters decreased with cryopreservation (p < .001) but did not differ between single and combined dual ejaculates. The number of insemination doses increased from 2.7 ± 0.4 for single to 4.7 ± 0.8 for combined dual ejaculates (p < .01), based on 100 million motile spermatozoa per frozen-thawed semen dose. In conclusion, combining two ejaculates collected at short interval for one cryopreservation process increases the number of AI doses without compromising semen quality.     

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.     

Sperm migration in pigs after deep intrauterine and intraperitoneal insemination

Deep intrauterine insemination in pigs allows sperm deposition only into one uterine horn, but bilateral fertilization of oocytes occurs. How the sperm reach the contralateral oviduct remains disputable. The aim of this experiment was to study possible transperitoneal and/or transuterine sperm migration ways. Follicle growth and ovulation were induced in 24 peripubertal gilts with eCG and hCG 72 h after eCG. Endoscopic intrauterine insemination (IUI) was performed 32 h after hCG with 20 ml of extended semen (60 × 10(6) spermatozoa) as follows: Group CONTROL (n=8) received IUI into the right horn, and the left horn served as non-treated control; Group LIGATURE (n=8) received IUI into the right horn, and the left horn was closed by endoscopic double ligature close to the bifurcation; Group INTRAPERITONEAL (IPI; n=8) received IUI into the right uterine horn, the left horn was closed by double ligature and semen was deposited intraperitoneally at the surface of the left ovary. Genital tracts were removed 65-66 h after hCG, the oviducts were flushed and ova (n=299) were analyzed for fertilization and cleavage. Furthermore, the accessory spermatozoa count/oocyte was graded as 0, without spermatozoa, 1, <5 spermatozoa, 2, 5-50 spermatozoa, 3, 50-100 spermatozoa and 4, >100 spermatozoa. The results indicate that low dose IUI into one horn provides a lower grade of accessory spermatozoa in the contra-lateral side (1.6 vs. 2.8). No spermatozoa were found in ova flushed from oviducts of the ligated uterine horn, even after intraperitoneal insemination (P<0.05), and no fertilization occurred, respectively. Our results clearly indicate that after low dose IUI into one uterine horn, spermatozoa reach the contralateral oviduct via transuterine migration.     

Genetic Parameters for Semen Production Traits in Austrian Dual-Purpose Simmental Bulls

Genetic parameters were estimated for semen production traits collected in an Austrian AI centre in the years 2000-2004. In total, 12,746 ejaculates from 301 Austrian dual-purpose Simmental (Fleckvieh) AI bulls were examined considering different effects on ejaculate volume, sperm concentration, percentage of viable spermatozoa in the ejaculate, total spermatozoa per ejaculate and motility. The model for genetic parameter estimation included the fixed effects age of bull, collection interval, number of collections on collection day, bull handler, semen collector, year and month of collection, a random additive genetic component and a permanent environmental effect. Correlations between estimated breeding values for semen traits and male fertility from the routine evaluation were calculated. The fertility trait considered in the routine evaluation is non-return rate 90 for the first insemination. All semen production traits were moderately heritable. Heritabilities for volume, concentration, percentage of viable spermatozoa, total number of spermatozoa and motility were 0.18, 0.14, 0.10, 0.22 and 0.04, respectively. Correlations between breeding values for semen quality traits and routinely estimated breeding values for male fertility were low and ranged from 0.08 to 0.17 indicating that semen production traits are rather poor predictors of male fertility.     

Low Dose Insemination in the Sow – A Review

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

Comparative study of different methods for dog semen cryopreservation and testing under clinical conditions

The extenders and freezing rates from three different freezing protocols were combined and compared to each other in order to study the post-thawing acrosome integrity and fertility of frozen dog sperm. A commercial bovine TRIS-base extender (TRILADYL) and two self-made canine semen extenders (Norwegian and Dutch) were combined with a conventional bovine and two canine freezing regimes, and acrosome integrity of frozen/thawed spermatozoa was assessed by fluorescein isothiocyanate conjugated peanut agglutinin staining (FITC-PNA). Differences between freezing/thawing protocols were reflected in the proportion of cells with acrosomal damage and not based on motility results. It was concluded that during dog semen cryopreservation extenders had less influence on the post-thawing sperm quality than did the freezing rates. The optimal extender/freezing rate combination (TRILADYL/Norwegian) was used in the clinical practice to evaluate the fertility of frozen sperm administered by intrauterine insemination using a surgical approach. The pregnancy rate was 57% (4/7), but the average litter size was low (2.8). This may have been due to the insufficient sperm numbers contained in an insemination dose and/or to the incorrect timing of artificial insemination (AI). The final conclusion is that the commercial bovine extender is useful for freezing dog semen, and the TRILADYL/Norwegian freezing protocol is recommended as the most advantageous combination for the freezing of canine semen in the clinical practice.