OC5Filtration Thought Sephadex Columns Does not Alter the Subpopulation Characteristics in Fresh Boar Semen

The aim of this study was to analyse the effect of filtration through Sephadex on the subpopulation characteristics of the boar semen. For this purpose 3 ml of 16 commercial doses of fresh diluted boar semen were filtered through a Sephadex G‐15/Polypropylene column. Motility parameters were analysed by a CASA system and statistical study was performed by SAS package using the VARCLUS and the FASTLUST procedures. Statistical study revealed four subpopulations in fresh boar semen, as previously had described (Theriogenology 61: 673–690).Total motility was higher in control than in filtered semen, but there were not statistical differences (65.63 ± 9.65 vs 41.40 ± 9.02). Moreover, the analysis did not show many changes neither in the characteristics nor in the distribution of the four subpopulations. As example although ALHmed of filtered samples were slightly higher, there were only significant differences (p < 0.001) in two subpopulations (subpopulation 2 : 2.2 ± 0.05 in control vs 2.7 ± 0.08 in filtered. Subpopulation 3 : 4.5 ± 0.11 in control vs 5.8 ± 0.23 in filtered semen). HME was also statistically different (p < 0.005) in one subpopulation, showing great values in filtered semen (1.7 ± 0.15 vs 3.0 ± 0.30). In conclusion, the filtration by Sephadex/Polypropylene column does not cause strong changes in subpopulation sperm distribution.     

OC4Influence of Seminal Plasma Added to Highly Diluted Semen on Sperm Motility of Young Boar Feed with Selenium and Vitamin E

High dilution rates have been documented as detrimental for boar spermatozoa, shortening their lifespan (Centurion et al. 2003, Biol Reprod 69: 640–646). Addition of seminal plasma (SP) to semen extenders, or selenium (Se) and vitamin E (VE) in diet of boars could increase motility of highly diluted spermatozoa (HDS). The aim of this work was to evaluate the effect of seminal plasma on sperm motility of HDS from boars feed with Se and VE. Sixteen 12 month-old boars were designed to one of four dietary treatments: (i) control, Se 0 ppm–VE 0 IU/kg; (ii) Se 0–VE 250; (iii) Se 0.5–VE 250 and (iv) Se 0.5–VE 0. Boars were treated for 8 weeks before semen collection. Sperm rich fractions from each boar were diluted to 5 × 10⁶ sperm/ml in PBS medium and incubated at 37°C with or without 10% SP. The measurements were done at 0, 2 or 5 h. Data were analyzed as a mixed model for a factorial design [2 (Se) × 2 (VE) × 2 (SP) × 3 (h)]. Percentage of sperm motility (PSM) increases significantly (p < 0.001) with addition of Se (81.3 ± 1.52), VE (81.0 ± 1.62) and SP (81.5 ± 1.57) vs control (73.4 ± 1.61). There was significant interaction Se × VE (p < 0.001) and Se × VE × SP (p < 0.05) in PSM. However, PSM was affected significantly by time (0 h 83.4 ± 1.92; 2 h 80.7 ± 1.92 and 5 h 67.9 ± 1.92; p < 0.001). There was significant interaction SP × Time (p < 0.05) in PSM. These results indicate that Se, VE and SP improve seminal viability. Addition of 10% of SP maintains PSM at least during 5 hours.     

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

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

Effects of Matrix Filtration of Low-Quality Boar Semen Doses on Sperm Quality

The aim of this work was to develop a method to enhance the sperm parameters of ejaculates with low sperm quality from Piétrain boars. Seminal doses were filtered through columns of DEAE Sephadex (length 2.5 ± 0.5 cm), CM Sephadex (length 5 ± 0.5 cm), glass wool (length 2 ± 0.5 cm) or glass bead (length 10 ± 0.5 cm), with an exit flow rate of 1 ml/40 s in all cases. For each male, 10 ml of the sperm cell-rich fraction diluted at 1 : 6 were filtered. Sperm quality was assessed before and after filtration. Sperm morphology, sperm motility and sperm concentration were determined using the computer program sca ^® 2002 Production, and sperm viability was evaluated by fluorescence multistaining. Osmotic resistance test and hyperosmotic resistance test were used to determine the osmotic resistance of spermatozoa, whereas l -lactate production estimated the metabolic activity. Results showed a decrease of sperm concentration and osmotic resistance of spermatozoa after filtration in the four matrixes. However, an increase in the frequency of viable spermatozoa with intact acrosome after filtration in glass bead columns and an increase of morphologically normal spermatozoa after filtration in Sephadex CM-50, glass wool and glass bead columns were observed. Despite the decrease in the frequency of progressive motile spermatozoa, l -lactate production and mitochondrial sheath integrity maintained constant after filtration. Our findings indicate that column filtration is an effective method to enhance the sperm quality by selecting viable and morphologically normal spermatozoa without altering DNA, plasma membrane, mitochondrial sheath integrity or inducing premature acrosome reaction.     

Seasonal Changes in Testicular Cell Morphology in Domestic Male Cats (Felis catus)

The aim of this study was to asses the variation in the morphology of the seminal epithelium in relation to natural photoperiod in male cats. Tom cats (n = 240) were castrated every other week throughout the year. Each testis was fixed in Bouin's solution and cut into sections. The percentage of tubules with round spermatids (RS), elongated spermatids (ES), tailed spermatids (TS), mature spermatids (MS) and the number of Sertoli cells (SC) and Leydig cells (LC) were recorded in each sample. Testicles from males during short days (SHD) had a higher percentage of tubules with RS and ES compared to testicles from males during long days (LHD, 31.3 ± 0.6 vs 2.1 ± 0.6%, p < 0.001; 30.9 ± 0.7 vs 11.0 ± 0.7%, p < 0.001). Conversely, testicles from males during SHD had a lower percentage of tubules with TS and MS compared to testicles from males during LHD (24.5 ± 0.8 vs 29.7 ± 0.8%, p < 0.01; 13.1 ± 1.2 vs 57.0 ± 1.2%, p < 0.01). Furthermore, testicles from males during SHD had a higher number of SC and lower number of LC compared to testicles from males during LHD (11.4 ± 0.1 vs 8.0 ± 0.1%, p < 0.01; 19.2 ± 1.0 vs 38.0 ± 1.0%, p < 0.01). In conclusion, there are seasonal changes in testis cell morphology in the tom which may be related to seasonal sperm production.     

An Experimental Model to Study Resistance Index and Systolic/Diastolic Ratio of Uterine Arteries in Adverse Canine Pregnancy Outcome

The aim of this study was to describe the changes in the resistance index (RI) and systolic/diastolic ratio ( S / D ) of the uterine arteries during mid-pregnancy abortion induction in the dog. Sixteen 30–35 day pregnant bitches were randomly assigned to either a pharmacological protocol to interrupt gestation (n = 8) or were used as untreated control group (n = 8). Doppler assessments of uterine arteries blood flow were carried out before the initiation of the protocol and then every other day up to abortion (treated group) or parturition (control group). All treated bitches aborted 6 ± 1.2 days after initiation of the treatment (while none of the non-treated bitches aborted). Pre-treatment RI and S / D did not differ between groups (p > 0.2) while average post-treatment indexes were (mean ± SD): 0.62 ± 0.1 vs 0.53 ± 0.1 (p < 0.01) and 2.96 ± 0.9 vs 2.23 ± 0.3 (p = 0.01), for the treated and non-treated group respectively. Correlations between days to abortion and RI or S / D were 0.75 (p < 0.01) and 0.79 (p < 0.01) and, −0.78 (p < 0.01) and −0.73 (p < 0.01) for the treated and non-treated groups respectively. In the treated group, correlations between serum progesterone (P₄) concentrations and RI and S / D were −0.76 (p < 0.01) and −0.59 (p < 0.01) respectively. It is concluded that, during induction of abortion, RI and S / D of uterine arteries progressively increased while P₄ decreased.     

Unilateral and Bilateral Vasectomy in the Dog: Alkaline Phosphatase as an Indicator of Tubular Patency

Contents The objective of this research was to use a model of unilateral and bilateral occlusion of the ductus deferens in the dog to study the use of alkaline phosphatase (AP) as an indicator of tubular patency. Seven healthy cross bred dogs weighing 10–15 kg BW with normal spermiogram and AP concentrations in semen were used. From each dog, three semen samples were obtained before (intact) and after right (unilateral) and left (bilateral) vasectomy. The AP concentrations were measured in duplicates by a colorimetric method in each of the three fractions (first, second (sperm-rich), third) of each ejaculate. In addition, a macroscopic and microscopic evaluation of each ejaculate was carried out to assure its quality. Data were analysed by least squares analysis of variance using SAS^®. In intact and unilateral vasectomized dogs, 96.6% of AP measured in semen corresponded to the second sperm-rich fraction whereas 1.53 and 1.83% corresponded to the first and third fractions respectively. Total AP concentrations (first and second and third fraction) in vasectomized dogs were lower than in intact animals (19.857 vs 2284.431 ± 4.347 UAL; p < 0.001). AP concentrations were much lower in bilateral than in unilateral vasectomized dogs (142 vs 39.572 ± 4.347 UL, p < 0.001). In summary, AP concentrations in semen can be used as an early indicator of unilateral or bilateral lack of patency of the epididymal and deferent ducts in the dog.     

Effect of Dietary Supplementation with Cod Liver Oil on Cold Shock and Freezability of Boar Semen

Contents
The effect of dietary supplementation with cod liver oil (CLO), which is rich in n-3 polyunsaturated fatty acids (PUFAs), on resistance to cold shock and on freezability of boar semen was investigated. Ejaculates from 29 fertile Norwegian Landrace boars, randomly divided into control (n = 15) and CLO-group (n = 14), were frozen before and after a 12 week period of daily oil supplementation. Before each freezing, semen samples were taken to determine the fatty acid composition of the spermatozoa. Docosahexaenoic acid (22 : 6n-3; DHA) was the major fatty acid in total lipids. The n-3 fatty acid DHA increased in the CLO-group from 25.5 to 32.1% at the expense of the n-6 docosapentaenoic acid (22 : 5n-6), which decreased from 11.3 to 4.2% (p < 0.0001). The concentration of these fatty acids were unchanged in the control group. There was also a significant decrease of other PUFAs in the CLO-group (p < 0.05). Eicosapentaenoic acid (20 : 5n-3) was not found in any sample. At four different steps of the preservation process (30, 15, 5°C and after freezing/thawing) both motility and acrosome integrity were assessed. No significant differences were found either within or between the groups at any of the steps. In conclusion, CLO-supplementation alters the lipid composition of the membranes of boar spermatozoa, however, this does not seem to have any beneficial effect on cold shock and freezability of boar semen.     

Recovery and Cryopreservation of Epididymal Sperm of Plains Bison (Bison bison bison) as a Model for Salvaging the Genetics of Wood Bison (Bison bison athabascae)

The objective of this study was to optimize recovery and cryopreservation of epididymal sperm from plains bison, as a model for wood bison. In Phase 1, cauda epididymides were recovered from bison (n = 14) immediately after slaughter, minced and incubated in Sp-TALPH buffer for 3 h at 36°C. The resulting sperm suspensions were cryopreserved in Triladyl^®, using a protocol for bovine semen. In Phase 2, epididymal sperm were cryopreserved in either Triladyl^® or Andromed^®. The mean (±SD) estimated number of sperm recovered was 468 ± 207 × 10⁶. There was an increase (p < 0.05) in the proportion of sperm with normal morphology between initial recovery and after extension (52.4 ± 4.6 vs 69.7 ± 2.4%), with a concurrent decrease (p < 0.05) in the proportion of sperm with distal droplets. Median values for progressively motile sperm in post-thaw samples (60%) were lower (p < 0.05) than that after extension or after chilling (70% for both). The mean percentages of viable sperm and of sperm with an intact acrosome were lower (p < 0.05) for frozen-thawed samples (38.7 ± 2.8 and 85.2 ± 1.1) compared with extended (66.2 ± 2.2 and 92.4 ± 0.9) or chilled (63.7 ± 2.5 and 90.0 ± 1.0) samples. Rates of cleavage, morulae and blastocyst production were not significantly different for chilled (70.9, 38.7 and 8.0%) vs post-thaw sperm (73.0, 46.0 and 6.3%). There was no significant difference between extenders for most sperm characteristics. In conclusion, we developed a functional protocol for the recovery and cryopreservation of epididymal sperm from plains bison, which may have implications for the genetic preservation of wood bison.     

Reproductive Performance of Gilts with Similar Age but with Different Growth Rate at the Onset of Puberty Stimulation

The aim of this study was to evaluate the reproductive performance of gilts that had a similar age but different weights at the onset of puberty stimulation by boar exposure at 144 days. Gilts were divided into two groups according to their lifetime growth rate from birth to approximately 144 days of age. Mean growth rates at this moment were 577 and 724 g/day for group 1 (G1; n = 58) and group 2 (G2; n = 58), respectively. After selection, gilts were weighed at approximately 155, 165 and 175 days of age, on the insemination day and at slaughter. Gilts were inseminated, on average, at 193 days of age and were slaughtered 32 days after insemination, when the number of corpora lutea and embryos were recorded. Higher growth rate gilts (G2) reached puberty earlier (155.3 vs 164.1 days; p < 0.01). More gilts of G2 group attained puberty by 190 days of age (p = 0.004) than G1 gilts (95%; 55/58 vs 76%; 44/58). The anoestrous rate, until 60 days after the onset of boar exposure was higher (p < 0.01) in G1 (19.0%; 11/58) than in G2 (3.4%; 2/58) group. However, there were no differences in the pregnancy rate (90.7 vs 94.5), ovulation rate (15.9 vs 16.5), total embryos (12.9 vs 11.7), viable embryos (12.0 vs 11.1) and embryo survival (73.7% vs 68.5%), between G1 gilts and G2 gilts, respectively (p > 0.05). High growth rate gilts attain puberty earlier and have a lower anoestrous rate than low growth rate gilts.     

Incubation of Post-Thaw Epididymal Cat Spermatozoa with Seminal Plasma

In domestic cats, epididymal spermatozoa have lower initial motility and viability than ejaculated spermatozoa and it is possible that seminal plasma compounds are behind these effects. The aim of this study was to investigate whether co-incubation of post-thaw epididymal cat spermatozoa with seminal plasma was able to improve sperm quality. Epididymal cat spermatozoa from 11 cats were cryopreserved. After thawing, each sperm sample was divided into two aliquots, centrifuged and incubated with two different media; Tris buffer (control) or pooled seminal plasma (treatment). Sperm quality was observed at 0, 2, 4 and 6 h after incubation. The results demonstrated that all of the sperm parameters except acrosome integrity were lower in the treatment group compared to the control group (p < 0.05); the percentages of motility (46.4 ± 15.4 vs 40.0 ± 9.4), the scores of progressive motility (3.1 ± 0.4 vs 2.8 ± 0.5), the percentages of spermatozoa with intact plasma membrane (46.3 ± 9.7 vs 39.6 ± 8.9) and intact acrosome (36.5 ± 16.2 vs 32.9 ± 15.1), as well as at all time points. In conclusion, the seminal plasma seems less beneficial to the post-thaw epididymal cat spermatozoa than the Tris buffer.     

Isolation and Culture of Ovine and Bubaline Small and Large Pre-antral Follicles: Effect of Cyclicity and Presence of a Dominant Follicle

Studies were conducted to examine the effects of the cyclicity and the presence of a dominant follicle (DF) in ovary on the recovery and in vitro growth of pre-antral follicles (PFs) in sheep and buffalo. Small pre-antral follicles (SPFs, 100–250 μm) and large pre-antral follicles (LPFs, 250–450 μm) were isolated from slaughterhouse ovaries in the breeding seasons by a mechanical and enzymatic method. The sheep and buffalo PFs were cultured in vitro for 6 and 15 days, respectively, and examined for their growth, survival and antrum formation rates and growth rates of oocytes in cultured pre-antral follicles. The follicles of the sheep and buffalo were recovered and cultured simultaneously within replicates. The recovery rates (number per ovary) of both SPFs and LPFs were significantly (p < 0.05) higher in cyclic ewes (SPFs: 22.0 ± 3.3 vs 12.1 ± 2.6 and LPFs: 16.0 ± 3.6 vs 9.2 ± 1.8) and buffaloes (SPFs: 9.2 ± 1.3 vs 4.1 ± 1.0 and LPFs: 10.3 ± 2.7 vs 5.4 ± 0.7) compared with those recovered from acyclic ones. Presence of a DF in ovary significantly (p < 0.05) reduced the recovery rates of LPFs in ewes (9.06 ± 2.7 vs 16.4 ± 3.8) but had no effect in buffalo. Cyclicity of animals or follicular dominance had no effects on in vitro growth, survival and antrum formation rates and growth rates of oocytes in cultured PFs of SPFs and LPFs in both sheep and buffalo. The in vitro growth, survival and antrum formation rates of LPFs and growth rates of oocytes in cultured LPFs were significantly (p < 0.05) higher than those observed in SPFs in both sheep and buffalo. The overall recovery and growth rates of the PFs were lower in buffaloes compared with ewes.     

Assessing in vivo Fertilizing Capacity of Liquid-Preserved Boar Semen According to the 'Hanover Gilt Model'

The goal of this study was to determine the ability of the Hanover gilt model to assess in vivo fertilizing capacity of preserved sperm and to consider whether any modifications to this model were needed. This model evaluates the fertilizing capacity of semen based on the fertilization rate, the rate of normal embryos and the accessory sperm count of 3–5‐day embryos. Its distinguishing characteristics are the use of one‐time insemination of sperm in reduced numbers, of spontaneously ovulating gilts and of ovulation detection through ultrasound examination of ovaries. Reduced sperm numbers allow for an accurate evaluation of the fertilizing potential of different semen treatments, thereby avoiding the compensatory effect of doses calibrated to maximize fertility. The model's usefulness was assessed in a trial run designed to compare the fertilizing capacity of liquid boar semen diluted into two different extenders. The diluent, the boar and the backflow, had no significant effect on any of the parameters studied. Gilts inseminated less than 24 h before ovulation had a significantly higher (p < 0.01) fertilization rate and accessory sperm cell count (p < 0.05) than those inseminated more than 24 h before ovulation. Very good/good embryos from homogeneous litters (only very good/good embryos were present) had a significantly higher (p < 0.01) accessory sperm count than those from heterogeneous litters (at least one embryo was of a different quality and/or oocytes were present). Both very good/good and degenerated/retarded embryos from heterogeneous litters had low accessory sperm numbers. This suggests that accessory sperm count is significantly related to the quality of the litter, but not to the quality of the embryo within gilts. It can be concluded that the Hanover gilt model is sensitive enough to show fertility differences (in this study, those associated with in vivo ageing of semen), while using relatively few gilts and little time.     

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

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

Effect of Oxytocin Treatment on Artificial Insemination with Frozen–Thawed Semen in Murciano–Granadina Goats

The site where the semen is deposited appears to be one of the most important factors affecting pregnancy of inseminated goats. In Murciano–Granadina (MG) goats, post-cervical insemination is achieved in a limited number of females. An effective way to increase fertility rate could be by increasing post-cervical inseminations. Effect of exogenous oxytocin application to facilitate the cervical penetration and its effect on kidding rate and prolificacy in MG goats were investigated. Oestrus was synchronized using progesterone-impregnated sponges for 11 days. Females were randomly divided into three groups (n = 190) and received either an i.v. injection of 100 or 200 IU of oxytocin or saline solution 15 min before being inseminated. Data on semen deposition depth were recorded for each animal using a catheter scaled in centimetres (up to 4 cm). Depth of semen deposition was affected by the oxytocin treatment (p < 0.05). Oxytocin enhanced cervical passage only with the dose of 200 IU compared with the control group, increasing the deposition depth (2.9 cm vs 1.9 cm). No significant effect of oxytocin treatment on kidding rate and prolificacy was detected. Depth of semen deposition affected kidding rate (p < 0.01). In conclusion, oxytocin treatment improved the depth of semen deposition in AI of MG goats, but kidding rate and prolificacy was not affected. More studies must be conducted to assess the minimal effective dose required for sufficient cervical dilation, and to determine the effects of such doses of oxytocin on uterine motility, sperm transport and fertility in goats.     

Sperm preparation through Sephadex™ filtration improves in vitro fertilization rate of buffalo oocytes

Routinely, swim‐up method is used to separate high‐quality sperm; however, long processing time and close cell‐to‐cell contact during the centrifugation step are inevitable elements of oxidative stress to sperm. The objective was to evaluate Sephadex^? and glass wool filtration to separate motile, intact and viable sperm for in vitro fertilization in buffalo. The cumulus–oocyte complexes (COC s) were collected from ovaries of slaughtered buffaloes by aspiration and matured for 24 hr in CO ₂ incubator at 38.5°C and 5% CO ₂. Matured COC s were rinsed twice in fertilization TALP and placed in the pre‐warmed fertilization medium without sperm. Cryopreserved buffalo semen was thawed at 37°C for 30 s and processed through Sephadex^?, glass wool filtration and swim‐up (control). Total and motile sperm recovery rates were assessed, resuspended in fertilization TALP and incubated for 15–20 min in CO ₂ incubator. Samples prepared by each method were divided into two aliquots: one aliquot was studied for sperm quality (progressive motility, membrane integrity, viability, liveability), while the other was subjected to co‐incubation with sets of 10–15 in vitro matured oocytes. Data on sperm quality were analysed by ANOVA , while in vitro fertilizing rates were compared by chi‐squared test using SPSS ‐20. Least significant difference (LSD ) test was used to compare treatment means. Glass wool filtration yielded higher total and motile sperm recovery rate, while Sephadex^? filtration improved (p  <  .05) sperm quality (progressive motility, membrane integrity, viability, liveability). Sperm preparation through Sephadex filtration yielded higher in vitro fertilization rate in terms of cleavage rate compared to glass wool filtration and swim‐up (control). In conclusion, cryopreserved Nili‐Ravi buffalo sperm selected through Sephadex filtration showed improved quality and yielded better fertilization rates (cleavage rate) of in vitro matured/fertilized oocytes. Sephadex filtration could be a promising technique for use in in vitro fertilization in buffalo.     

Identification of Sperm Subpopulations in Stallion Ejaculates: Changes after Cryopreservation and Comparison with Traditional Statistics

In an attempt to improve the information obtained after computer-assisted sperm analysis (CASA), data from five stallions (three ejaculates from each) were analysed before (fresh, extended semen) and after cryopreservation using traditional statistics as well as a cluster analysis. The data matrix consisted of 13 987 observations of individual spermatozoa for fresh, extended semen, and 8305 for frozen–thawed samples. As expected, freezing and thawing resulted in a marked decrease of CASA-derived variables of sperm kinematics. All sperm velocities were significantly lower in frozen–thawed samples than in samples before cooling. Using sperm velocities, six sperm subpopulations were identified in fresh semen (S1–S6). As such, subpopulations S1 and S2 were characterized by low sperm velocities, subpopulations S3 and S4 corresponded to spermatozoa depicting medium speed values, and finally, subpopulations S5 and S6 were those depicting the highest velocities. After freezing and thawing, four sperm subpopulations were identified, listed as nr FT1 to FT4. While subpopulations FT1–FT3 were characterized by low sperm velocities, and thus corresponded speed-wise to those listed as S1–S4 for fresh, extended semen, the one called number FT4 in frozen semen was characterized by high velocities, of the same range as that of the subpopulations S5 and S6 for fresh spermatozoa. The sperm subpopulation structure varied among stallions, but the cluster analysis hereby assayed was able to provide valuable information about the freezability of the samples that the customary statistics did not reveal.     

Concentration,Activity and Biochemical Characterization of Myeloperoxidase in Fresh and Post‐Thaw Equine Semen and their Implication on Freezability

Myeloperoxidase (MPO) is a pro‐oxidant enzyme associated with decreased motility in thawed equine semen. This study aimed to describe MPO concentration, activity and subunits in raw and thawed semen and to correlate these data with motilities in raw and thawed semen. Semen samples from five stallions were collected four times. Motilities were assessed in raw and thawed semen. MPO assays were performed in raw seminal plasma, raw sperm‐rich pellet and thawed semen. Total and active MPO concentrations were, respectively, assayed by enzyme‐linked immunosorbent assay and specific immunological extraction followed by enzymatic detection. MPO subunits present in semen were characterized by Western blot. Purified active MPO was added in saline solution and freezing extender to control its activity during freezing procedure. Differences between medians were determined using Kruskal–Wallis test, and correlations were determined using Spearman's test for nonparametric data. Active MPO concentration was low in seminal plasma and thawed semen, but high in pellet (p = 0.0058), as the opposite relation was observed for total MPO concentration (p < 0.0001). In seminal plasma and post‐thaw semen, inactive 86‐kDa MPO precursor was mainly observed. Purified MPO activity was decreased in the extender (p = 0.0286). MPO activity in pellet was highly correlated with thawed progressive motility (r = ?0.5576, p = 0.0086). Inactive MPO precursor and unknown low molecular weight inactive MPO precursor subunits explain low MPO activity in semen. Major MPO activity was observed in pellet, and post‐thaw loss of activity is partially explained by MPO inactivation in extender. Thawed semen motility was negatively correlated with MPO activity in pellet, becoming a potential freezability predictor.     

Reproductive Parameters and Efficiency of Inseminators in Dairy Farms in Portugal

This study was carried out to determine the reproductive efficiency indices of one of the largest dairy co-operatives of northern Portugal, using data from 1980 to 1998. Records were made available by the computerized National Recording System. Age at first calving was 32.0 ± 6.0 months. Mean calving to first AI interval was 95.4 ± 30.0 days, and calving to conception intervals decreased (p < 0.05) from 176.9 ± 4.5 to 148.1 ± 5.6 days from the first to the fourth/fifth parturitions, respectively. Calving intervals decreased (p < 0.05) from 418.1 ± 3.4 to 392.5 ± 7.0 days from the first to fourth/fifth parturitions, respectively. Mean non-return rates at 90 days for first inseminations was 71.7 ± 6.5% and mean calving rates at first insemination was 51.4 ± 8.1%. There were significant differences (p < 0.001) in the inseminators' efficiency, measured by both non-return and calving rates at first AI, with differences between the best and worst results of 13.3 and 16.1% for non-return and calving rates, respectively. The ranking of the inseminators did not coincide when their efficiency was measured by either non-return or calving rates. The mean number of inseminations per pregnancy (pregnant cows only) was 1.4 ± 0.7 with significant (p < 0.001) differences among herds. The mean heat detection rate was 38.1 ± 16.9%, with highly significant (p < 0.001) differences among farms (ranging from 14.2 to 60.8%). Negative (p < 0.001) correlations were found between heat detection rate and calving to first AI, calving to conception and calving intervals. The meaning of these indexes for assessment of reproductive efficiency in the studied system, is discussed.     

Expression of MHC-I and -II in Uterine Tissue from Early Pregnant Bitches

The aim of the study was to investigate the expression of major histocompatibility complex (MHC)-I and -II in uterine tissues from pregnant and non-pregnant bitches, taken at different time periods after mating. The pregnant bitches were ovariohysterectomized during the pre-implantation (group 1, n = 4), implantation (group 2, n = 7) and placentation stage (group 3, n = 7). Non-pregnant animals in diestrus served as controls (group 4, n = 7). The expression of MHC- I and -II in salpinx, apex, middle horn, corpus uteri and at implantation sites was investigated by immunohistochemistry as well as qualitative and quantitative RT-PCR; MHC-I mRNA was detected in all tissues and with quantitative RT-PCR, and no significant changes were detected until placentation. Immunohistologically, at the apex and corpus site, the average number of MHC-II positive cells increased from the pre-implantation to the post-implantation stage (apex: 1.54 ± 1.21 to 3.82 ± 2.93; corpus: 1.62 ± 1.9 to 5.04 ± 4.95; p < 0.05). The greatest numbers of MHC-II positive cells were observed at placentation sites (6.64 ± 5.9). In parallel, a marked increase in the relative mRNA expression of MHC-II in uterine tissues was assessed from the pre-implantation to the placentation stage (relative to Glycerinaldehyd-3-phosphate-Dehydrogenase (GAPDH): 6.9 ± 9.5, 8.4 ± 5.8, p > 0.05). Immunohistologically, in the salpinx, significantly greater numbers of MHC-II positive cells were found in the tissues of pregnant animals than in the control group (p < 0.05). It is proposed that the increase in MHC-II is pregnancy-related, even though the impact on maintenance of canine pregnancy is still unclear.