Biomedical subjects

A Van Soom

Publications and source records attributed to A Van Soom.

At least 37 records · Page 2Linked to original sources

PubMed2004

Effect of non-esterified fatty acids on bovine granulosa cell steroidogenesis and proliferation in vitro.

In high-yielding dairy cows, the negative energy balance (NEB) during the first weeks post partum may influence dominant follicle growth and steroidogenesis. Since non-esterified fatty acid (NEFA) concentrations are elevated during NEB and are shown to be toxic for several cell types, we investigated the individual and combined effects of the three main NEFA's on granulosa cell proliferation and steroidogenesis in vitro. Granulosa cells from large follicles were cultured for two days in serum free medium in the presence of palmitic (C16:0) (PA), stearic (C18:0) (SA) and/or oleic acid (C18:1) (OA). Addition of 150, 300 or 500 microM of PA and SA inhibited cell proliferation (P<0.05) while OA only elicited such an effect at 500 microM (P<0.01). In the combination treatment (150 microM of each fatty acid), cell numbers were also reduced (P<0.01). These inhibitory effects on cell number are partly due to the induction of apoptosis by these NEFA's, as was demonstrated by annexin V-FITC/propidium iodide staining of the granulosa cells. Oestradiol-17beta production was stimulated by all doses of PA, by 300 and 500 microM of SA and by 500 microM of OA (P<0.05). Combined treatment with 150 microM of each fatty acid also stimulated oestradiol-17beta production per 10(4) cells (P<0.05). We can conclude that PA, SA and to a lesser degree OA modulate granulosa cell proliferation and steroidogenesis in vitro. These effects may be involved in the occurrence of ovarian dysfunction during the postpartum period in high-yielding dairy cows.

Animals↗

PubMed2004

Metabolic changes in follicular fluid of the dominant follicle in high-yielding dairy cows early post partum.

Characteristics of the intrafollicular environment to which the preovulatory oocyte is exposed may be one of the major factors determining subsequent fertility. The aim of our study was to examine to what extent metabolic changes that occur in early post partum high-yielding dairy cows are reflected in the follicular fluid (FF) of the dominant follicle (>8 mm). Nine blood samples were taken per cow from nine high-yielding dairy cows between 7 days before and 46 days after parturition. From Day 14 post partum on and together with blood sampling, FF samples of the largest follicle were collected from the same cows by means of transvaginal follicle aspiration. Serum and FF samples were analyzed using commercial clinical and photometric chemistry assays for glucose, beta-hydroxybutyrate (beta-OHB), urea, total protein (TP), triglycerides (TG), non-esterified fatty acids (NEFA) and total cholesterol (TC). All cows lost body condition during the experimental period (0.94+/-0.09 points) illustrating a negative energy balance during the experimental period. In FF, glucose concentrations were significantly higher and the TP, TG, NEFA and TC concentrations were significantly lower than in serum (P<0.05). The concentrations of glucose, beta-OHB, urea and TC in serum and in FF changed significantly over time (P<0.05). Throughout the study, changes of all metabolites in serum were reflected by similar changes in FF. Especially for glucose, beta-OHB and urea the correlations were remarkably high. The results from the present study confirm that the typical metabolic adaptations which can be found in serum of high-yielding dairy cows shortly post partum, are reflected in follicular fluid and, therefore, may affect the quality of both the oocyte and the granulosa cells.

3-Hydroxybutyric Acid↗

PubMed2004

A comparison of a mechanical sector and a linear array transducer for ultrasound-guided transvaginal oocyte retrieval (OPU) in the cow.

A comparison was made between a linear array and a mechanical multiple angle sector (MAP) transducer for ultrasound-guided transvaginal oocyte retrieval (ovum pick-up, OPU) in the cow. The ovaries of five dairy cows were punctured, in a twice-weekly OPU program lasting for 4 weeks, using two different 5.0-MHz transducers equipped with an identical disposable needle-guidance system. Both ovaries were visualized using each transducer before puncture and the number of follicles with a diameter of less than 5 mm (small) and with a diameter equal to or greater than 5 mm (large) was recorded. Subsequently, one ovary of the pair was punctured guided by the MAP, while the other was punctured using the linear array transducer. During the next puncture session on a given animal, the two systems were switched and used on the alternate ovary in a crossover design. Parameters assessed for each system were: the total number of follicles visualized in each diameter class, and the total number of retrieved oocytes per cow. A significant difference was found for the ability to visualize smaller follicles in favor of the MAP transducer, with an average visualization of 71.6 +/- 30.3 small follicles per cow during the 4-week trial period, compared to 59.8 +/- 25.7 for the linear array transducer (t-test for paired samples, P = 0.007). No differences were found in the visualization of large follicles. A numerically greater number of oocytes were retrieved using the MAP transducer, compared to the linear array, (averages of 14.2 +/- 7.2 versus 7.4 +/- 6.1, respectively), although these differences were not statistically significant. In conclusion, both systems can be effectively used for oocyte retrieval in the cow, however, the MAP transducer demonstrated superior visualization of small follicles.

Animals↗

PubMed2004

Susceptibility of pig embryos to porcine circovirus type 2 infection.

The aim of the present study was to determine if porcine circovirus type 2 (PCV2) is able to infect embryonic cells of in vivo produced porcine embryos with and without zona pellucida (ZP). ZP-intact and ZP-free morulae (6-day post-insemination) and early blastocysts (7-day post-insemination), and hatched blastocysts (8-day post-insemination) were exposed to 10(5.0) TCID50 PCV2 per ml (strain 1121, fifth passage PK15). At 48 h post-incubation, the percentage of infected embryos and the percentage of viral antigen-positive cells per embryo were determined by indirect immunofluorescence (IF). Significantly different percentages of infected embryos were detected: 15% for ZP-free morulae, 50% for ZP-free early blastocysts and 100% for hatched blastocysts. The percentage of cells that expressed viral antigens was similar for the three stages of development. PCV2 exposure did not affect the in vitro development of the embryos during the 48 h study period. All ZP-intact embryos remained negative for viral antigens. In an additional experiment the diameter of the channels in the porcine ZP was determined. After incubation of early blastocysts with fluorescent microspheres of three different sizes, beads with a diameter of 20 nm and beads with a diameter of 26 nm crossed the zona whereas beads with a diameter of 200 nm did not. In conclusion, it can be stated that PCV2 is able to replicate in in vivo produced ZP-free morulae and blastocysts and that the susceptibility increases during development. The ZP forms a barrier to PCV2 infection, but based on the size of the channels in the ZP the possibility that PCV2 particles cross the ZP cannot be excluded.

Animals↗

PubMed2004

Inhibition of bovine sperm-oocyte fusion by the p-aminophenyl derivative of D-mannose.

Several steps in the process of mammalian fertilization are mediated by carbohydrates. This study investigated the role of the p-aminophenyl derivative of d-mannose (APMP) during bovine fertilization. Inseminating cumulus-oocyte complexes (COCs) in the presence of increasing APMP concentrations resulted in a significant dose-dependent decrease of the fertilization rate (P < 0.05). No negative effect of 50 mM APMP on total sperm motility and progressive motility was found. Subsequently, the fertilization steps at which this blocking effect could be exerted were investigated, i.e., sperm penetration of the cumulus oophorus, sperm-zona binding, acrosome reaction, sperm-oolemma binding, and/or sperm-oocyte fusion. Inseminating cumulus-enclosed and cumulus-denuded oocytes in the presence of 50 mM APMP significantly decreased the fertilization rate to a comparable minimum level (P < 0.05). There was no significant relationship between the number of spermatozoa bound to the zona pellucida and the APMP concentration, and APMP nor d-mannosylated bovine serum albumin (BSA) suppressed or stimulated sperm acrosomal status. Inseminating zona-free oocytes in the presence of 50 mM APMP did not influence sperm-oolemma binding, but significantly inhibited sperm-oocyte fusion (P < 0.05). Preincubating zona-free oocytes with 200 microg/ml Con A but not with 50 mM APMP inhibited the sperm-oocyte fusion rate to the same extent as when the gametes were simultaneously exposed to 50 mM APMP. These data indicate that the blocking effect of APMP on bovine fertilization is mainly due to an inhibition of sperm-oocyte fusion, probably by specific obstruction of the sperm receptor sites that are responsible for the fusion process.

Acrosome Reaction↗

PubMed2004

[Deep intrauterine insemination in cattle].

INTRODUCTION: Most of the artificial inseminations in cattle nowadays are being performed in the uterine body with a rigid insemination device. Uterotubal junction insemination can only be performed in cattle with a device which is rigid enough to pass the cervix and flexible enough to follow the curvature of the uterine horns. At the Faculty of Veterinary Medicine in Ghent, a new insemination device has been developed for semen deposition near the utero-tubal junction in cattle and other animals. MATERIALS AND METHODS: In a first field trial the feasibility of the newly developed Ghent device was evaluated. Four thousand sixty-four dairy cows were inseminated by 12 inseminators with a standard insemination dose (10-15 million of frozen-thawed spermatozoa). Three insemination methods were compared; group 1: insemination in the uterine body with the conventional insemination device, group 2: insemination in the uterine body with the Ghent device, and group 3: insemination in the tip of both uterine horns with the Ghent device. In a second field trial insemination of dairy cows with the Ghent device was compared with the conventional insemination technique to evaluate the effect on pregnancy rates. The insemination dose was lowered to eight million (trial 1), four million (trial 2), and finally to two million frozen-thawed spermatozoa (trial 3). In each field trial, cows were divided into three groups: the first group was inseminated with a full insemination dose (12 x 10(6)) in the uterine body with the conventional insemination device, the second group with a lowered insemination dose in the uterine body with the conventional insemination device, and the third group with a lowered insemination dose in the tip of both uterine horns with the Ghent device. It can be concluded that decreasing the insemination dose from 12 to four million frozen-thawed spermatozoa had no effect on pregnancy rate in our experiments, neither with the conventional insemination device, nor with the Ghent device. The device is made of disposable materials and has been tested to be non-toxic for bovine spermatozoa, can be used by one person and is adapted for application in the field. CONCLUSION: In the near future, similar field trials will be performed with even lower doses of semen. It is only in these cases that we truly hope to show a positive effect of uterotubal junction insemination by using low quality semen or by using sexed semen.

Animals↗

PubMed2004

Metabolite and ionic composition of follicular fluid from different-sized follicles and their relationship to serum concentrations in dairy cows.

Metabolic changes in blood serum may be reflected in the biochemical composition of follicular fluid and could indirectly influence oocyte quality. The purpose of this study was to examine the biochemical composition of follicular fluid harvested from different-sized follicles and its relationship with that of blood serum in dairy cattle. Following slaughter, blood samples were collected from dairy cows n=30 and follicular fluid aspirated from three size classes of non-atretic follicles (<4 mm, 6-8 mm and >10 mm diameter). Samples remained independent between cows and between size classes within cows. Serum and follicular fluid samples were assayed using commercial clinical and photometric chemistry assays for ions (sodium, potassium and chloride) and metabolites (glucose, beta-hydroxybutyrate (beta-OHB), lactate, urea, total protein, triglycerides, non-esterified fatty acids (NEFA) and total cholesterol). Results showed that follicular fluid concentrations of glucose, beta-OHB and total cholesterol increased from small to large follicles and decreased for potassium, chloride, lactate, urea and triglycerides. There was a significant concentration gradient for all variables between their levels in serum and follicular fluid (P<0.05). Significant correlations were observed for chloride (r=0.40), glucose (r=0.56), beta-OHB (r=0.85), urea (r=0.95) and total protein (r=0.60) for all three follicle size classes and for triglycerides (r=0.43), NEFA (r=0.50) and total cholesterol (r=0.42) for large follicles (P<0.05). The results from the present study suggest that the oocyte and the granulosa cells of dairy cows grow and mature in a biochemical environment that changes from small to large follicles. Furthermore, the significant correlation between the composition of serum and follicular fluid for the above-mentioned metabolites suggests that metabolic changes in serum levels will be reflected in the follicular fluid and, therefore, may affect the quality of both the oocyte and the granulosa cells.

3-Hydroxybutyric Acid↗

PubMed2004

Comparative study on five different commercial extenders for boar semen.

Increasing interest in a longer preservation of diluted boar sperm raises questions in the field concerning the choice of the extender. The aim of this study was to evaluate the longevity of boar sperm extended in currently used commercial semen extenders. Three long-term extenders and two short-term extenders were compared for different semen quality parameters that can be assessed under routine laboratory conditions. Sperm morphology, motility, pH and bacteriological contamination were investigated during a 7-day period. The number of dead spermatozoa did not differ significantly among the extenders (p > 0.05). Sperm motility was not only related with storage period but most of all with pH, especially in long-term extenders. Differences between the different extenders were prominent (p < 0.05); the sperm preserved in only one long-term extender showed good motility during the whole test period. In all cases, the pH of the extended semen increased by 0.3-0.5 in the first days of storage and was significantly correlated with a decrease in motility. Bacteriological quality had no significant influence on motility or pH of the semen. In conclusion, we can state that in both short-term extenders and in only one long-term extender, sperm longevity, as evaluated by the parameters used in this study, was sufficient during the preservation period. To preserve the quality of diluted boar semen during long-term storage, the choice of the long-term extender is important. In addition, the monitoring of the pH of extended boar semen in our study emphasizes the importance of the buffering capacity of semen extenders.

Animals↗

PubMed2004

Mapping and transcription profiling of CASP1, 3, 6, 7 and 8 in relation to caspase activity in the bovine cumulus-oocyte complex.

So far 12 caspases have been described in mouse and human while only one (CASP13) is known in cattle. The aim of this study was to (1) search for other bovine caspases by reverse transcription and polymerase chain reaction (RT-PCR) and (2) examine the presence of bovine caspase mRNA and active protein in the cumulus-oocyte complex. Five caspases (1, 3, 6, 7 and 8) were identified, partially cloned and sequenced. Four of them were mapped. Differential transcription of the caspase genes was detected, but no active caspase protein was found in diverse bovine oocytes. Cumulus granulosa cells (CGC) contain CASP1, 6, 7 and 8 mRNA and active caspase protein. The presence of caspase mRNA and active caspase proteins in CGCs suggests the occurrence of apoptosis in cumulus-oocyte complex, while caspase activation is blocked in fresh oocytes and therefore caspase transcription cannot be used to predict the oocyte developmental capacity.

Animals↗

PubMed2004

Intra-uterine insemination in farm animals and humans.

Artificial insemination (AI) is the oldest and currently most common technique in the assisted reproduction of animals and humans. The introduction of AI in farm animals was forced by sanitary reasons and the first large-scale applications with a commercial goal were performed in cattle in the late 1930s of last century. After the Second World War, cryopreservation of semen facilitated distribution and AI was mainly performed for economic reasons, especially in dairy cattle industry. In humans however, AI was initially performed in cases of physiological and psychological sexual dysfunction, but later on also in cases of infertility caused by immunological problems. Currently, the most common indications for intra-uterine insemination (IUI) in humans are unexplained infertility and male subfertility. In these cases, IUI is considered as the treatment of the first choice, before more invasive techniques such as in vitro fertilization (IVF) and intracytoplasmatic sperm injection (ICSI) are used. In contrast with humans, the quantity and quality of semen produced by farm animals is much higher and permits dilution and production of several insemination doses per ejaculate. However, with the introduction of sex-sorted semen in farm animals, the same problem of low-quality semen as in humans has arisen. In cattle, pigs and horses, conventional insemination with low numbers of sex-sorted spermatozoa results in a significant decrease in fertility. To improve the fertility rates with this semen, new insemination techniques have been developed in order to deposit spermatozoa closer to the site of fertilization. In sows and mares the advantage of utero-tubal junction (UTJ) insemination has already been proven; however, in cattle it is still under investigation. In this review, the differences and similarities in the application of AI between animals and humans are discussed and as AI in farm animals is most successful in cattle, the situation in this species is elaborated the most.

Animals↗

PubMed2004

Storage of fresh bovine semen in a diluent based on the ionic composition of cauda epididymal plasma.

For artificial insemination (AI) in cattle, much lower insemination doses can be applied when fresh semen is used instead of frozen-thawed semen. However, a particular disadvantage of fresh semen is its limited shelf life. As bovine spermatozoa can be stored for several weeks in the cauda epididymis without negative effects on their fertilizing capacity, it is an interesting organ to serve as a model in order to prolong the shelf life of fresh semen. First, the storage capacity of a diluent [cauda epididymal plasma (CEP-1)] with the same ionic composition, pH and osmolarity as the bovine CEP was compared with a Tris diluent for extended preservation of fresh ejaculated bovine semen. Secondly, the ionic composition of the CEP-1 diluent was modified (CEP-2) and its storage capacity was compared with this of the CEP-1 and Tris diluent. Finally, the effect of addition of different polyols (sorbitol, glycerol, mannitol) and egg yolk concentrations (5, 10 and 20%) to the CEP-2 diluent was assessed. Sperm quality decreased rapidly in the CEP-1 diluent. The quality and especially progressive motility of spermatozoa stored in the CEP-2 diluent were better those in the CEP-1 and Tris diluent. No significant effects of different sugars or egg yolk concentrations on the quality of fresh bovine semen in the CEP-2 diluent were observed. In conclusion, the CEP-2 diluent with 10% egg yolk and 1 g/l sorbitol may be used for extended preservation of fresh bovine semen at 5 degrees C up to 6 days.

Animals↗

PubMed2004

Motility assessment of porcine spermatozoa: a comparison of methods.

Although widely used in practice, visual motility assessment of boar spermatozoa is a very subjective method. The aim of this study was to compare the visual motility assessment of boar spermatozoa with two objective, automated systems, namely the Sperm Quality Analyzer (SQA-IIC) and the Hamilton-Thorne computer-based semen analyzer (HTR). In addition, concentrations as determined by the Burker counting chamber and HTR were compared. Motility of 30 semen samples from 30 different boars (22 Pietrain, seven Landrace boars and one Large White) was examined during three consecutive days, subjectively by two independent persons (visual motility assessment) and objectively with both automated systems. The use of the SQA-IIC and HTR for assessing boar sperm motility was evaluated and the repeatability of the measurements was estimated. The Sperm Motility Index (SMI), determined by SQA-IIC, and the percentage motile spermatozoa determined by the HTR showed a good correlation (r=0.71; p <0.05). The visual examination performed by Person 2 showed a good correlation with the SMI (r=0.81) and with the percentage of motile spermatozoa measured by the HTR (r=0.66) (p <0.05). There was a very poor correlation and a limited agreement between the visual assessments of both persons emphasizing the subjectivity of visual motility assessment. Nevertheless, motility scores of each person during the three consecutive days were highly correlated (r=0.67 and 0.72, p <0.05). The limits of agreement plots showed poor agreement between both persons and the HTR. The repeatability of measurements for most parameters evaluated by the HTR and by the SQA-IIC was good with coefficients of variation below 10%. In addition, for fertile Pietrain boars (n=22), reference values for the different HTR-parameters are presented showing a high curvilinear velocity (157.3 +/- 19.5 microm/s) and a low straightness and linearity of the movement of the spermatozoa (62.7 +/- 8.7 and 35.5 +/- 7.6%, respectively). Concentration as determined by the Burker counting chamber (56.0 +/- 16.8 x 10(6)/ml) was significantly higher compared with HTR measurement (37.6 +/- 7.7 x 10(6)/ml). The high number of counted cells and the low variation render the HTR concentration measurement more reliable. It can be concluded that visual motility assessment is highly subjective and should therefore be replaced by automated systems that allow for a more objective and detailed motility assessment of boar spermatozoa. In addition, based on the present results, highly repeatable results were obtained by the SQA-IIC and especially by the HTR.

Animals↗

PubMed2004

Sperm distribution in the genital tract of the bitch following artificial insemination in relation to the time of ovulation.

In the present study, sperm distribution in the genital tract of the bitch following artificial insemination (AI) in relation to the time of ovulation was investigated by histology, scanning electron microscopy (SEM) and flushing. Ten bitches were inseminated intravaginally with 500 x 10(6) spermatozoa: three dogs before ovulation, four dogs during ovulation and three dogs after ovulation. Ovariohysterectomy was performed 24 h after AI. Half of the genital tract was divided into nine segments (cervix, corpus uteri, caudal, middle and cranial uterine horn (UTH), utero-tubal junction (UTJ), isthmus, ampulla and infundibulum), which were processed for histology and SEM. The contralateral UTH and uterine tube (UT) were flushed, and several sperm characteristics were assessed. Histology revealed that the spermatozoa were mainly located in the uterine glands and at the UTJ, while very few spermatozoa were detected in the UT. Insemination during ovulation resulted in higher percentages of glands with spermatozoa in the different parts of the uterus (P < 0.05). Evaluation by SEM showed higher numbers of spermatozoa in several parts of the uterus for bitches inseminated during ovulation (P < 0.05). The mean number of spermatozoa flushed from the UTH and the UT was low. No significant differences in the evaluated sperm quality parameters were found between the flushings of the UTH and the UT. In conclusion, based on our findings, the uterine glands and the UTJ might act as sperm reservoirs in the bitch and sperm transport in the genital tract is affected by the time of AI in relation to ovulation.

Animals↗

PubMed2003

Influence of oxygen tension on apoptosis and hatching in bovine embryos cultured in vitro.

Various oxygen tensions are employed for in vitro embryo production. Since it is known that oxygen tension can influence the efficiency of embryo production and embryo quality, the aim of our study was to define an optimal oxygen concentration for bovine embryo production in vitro in synthetic oviduct fluid (SOF). Embryo quality criteria were hatching ability and the degree of apoptosis as assessed by TUNEL staining and Bax gene expression. In Experiment 1, the effects of 2, 5 and 20% O(2) tensions on embryo development were compared. The highest rate of eight-cell embryos (47%) at 72 hpi was obtained under 20% O(2). However, it seemed that 2 and 5% O(2) were also suitable as assessed by embryo survival rates at 144 hpi (29 and 30% at morula stage), 168 hpi (21 and 19% at blastocyst stage) and 216 hpi (14 and 17% at hatched blastocyst stage). In Experiment 2, comparisons were made between effects of 5, 20% and alternating O(2) (20% O(2) to 72 hpi and then changed to 5% O(2) up to 216 hpi) on embryo development. Alternating the O(2) tension significantly reduced the number of hatching blastocysts to 7%. Staining with TUNEL revealed that apoptosis occurred in all tested hatched blastocysts, but a significantly lower apoptotic cell ratio was found in embryos cultured under 5% O(2) (P<0.05). Total cell number of embryos cultured under 5% and alternating oxygen was significantly higher than that of other groups (P<0.05). Bax gene expression was detected by means of RT-PCR in only 2 of 66 hatched blastocysts. It can be concluded that 5% oxygen is optimal for bovine embryo culture in cell free media. Moreover, it is very likely that the apoptosis detected by TUNEL staining in this study is Bax-independent.

Animals↗

PubMed2003

Effect of sperm coating on the survival and penetrating ability of in vitro stored bovine spermatozoa.

The aim of this study was to examine the effect of sperm coating on the survival and penetrating ability of in vitro stored diluted spermatozoa. Bovine semen was collected by means of an artificial vagina connected with a tube containing 5 ml of the commercial Triladyl diluent supplemented with 20% egg yolk and 6.7% glycerol (EYTG). Both EYTG and seminal plasma were removed by centrifugation and the spermatozoa were stored under different in vitro storage conditions. In the first and second experiment, "control" and "coated" spermatozoa were stored in Hepes-TALP (pH 6 and 7) at room temperature. After 4 days of storage, the progressive motility, membrane integrity, mitochondrial membrane potential or DNA integrity of the spermatozoa were evaluated before and after Percoll centrifugation. The in vitro penetration rate of the spermatozoa was examined only after Percoll centrifugation. A significantly (P<0.05) positive influence of sperm coating was observed on the tested sperm characteristics and penetration rate of spermatozoa when they were stored in Hepes-TALP at pH 7, but not at pH 6. In the last experiment, the influence of the storage medium Hepes-TALP (pH 7) or EYTG was investigated on motility, membrane integrity, mitochondrial membrane potential and in vitro penetration potential of "coated" spermatozoa stored at room temperature or at 4 degrees C during 4, 5 and 6 days. After 6 days of storage, a significantly (P<0.05) higher percentage of motile and membrane intact spermatozoa with high mitochondrial membrane potential was obtained in EYTG at both temperatures leading to a significantly higher in vitro penetration rate. These results indicate that sperm coating could preserve sperm characteristics and penetrating capacity of fresh bovine spermatozoa stored in egg yolk containing diluent for up to 6 days.

Animals↗

PubMed2003

In vitro survival of bovine spermatozoa stored at room temperature under epididymal conditions.

In this study, environmental conditions mimicking those prevailing in the epididymis were used for storing ejaculated bull spermatozoa in vitro during 4 days at ambient temperature. These conditions were low pH, high osmolarity, high sperm concentration and low oxygen tension. Hepes-TALP was used as basic storage medium. Fresh spermatozoa were stored at a concentration of 10 x 10(6)spermatozoa/ml in Hepes-TALP of different pH (pH 4, 5, 6, 7 or 8), and osmolarity (100, 300, 400, 500, 600 or 800 mOsm/kg), and under different atmospheric conditions (nitrogen gassed or aerobic). Spermatozoa were also stored undiluted or at different concentrations: 10x 10(6), 100 x 10(6), 500 x 10(6) or 1 x 10(9)spermatozoa/ml. Sperm parameters such as membrane integrity, motility, mitochondrial membrane potential or DNA fragmentation were used to assess semen quality after storage. Adjustment of the pH of Hepes-TALP to pH 6 yielded significantly better results than storage at all other pH values. Isotonic Hepes-TALP (300 mOsm/kg) had a less detrimental effect on spermatozoa than hypo- and hyperosmotic versions. No differences in sperm parameters were observed when spermatozoa were incubated under aerobic or under nitrogen gassed storage conditions. Optimal sperm concentration in vitro is 10 x 10(6)spermatozoa/ml. This is in contrast with the in vivo situation, where spermatozoa are stored at high concentration. However, better results at high sperm concentrations were obtained when spermatozoa were diluted for less than 5 min in Triladyl-egg yolk-glycerol diluent immediately after ejaculation.

Animals↗

PubMed2003

Motility characteristics of boar spermatozoa after addition of prostaglandin F2alpha.

Addition of prostaglandin F2alpha (PGF2alpha) to extended boar semen has been shown to slightly increase reproductive parameters in sows such as the conception rate and the total number of piglets born alive. The mechanisms by which PGF2alpha affect these parameters have not yet been elucidated, but it is possible that the sperm transport after insemination is increased. This study investigated whether the sperm motility from 20 Piétrain boars improved when PGF2alpha (Dinolytic; 5 mg PGF2alpha/ml) was added to diluted semen. Different amounts of PGF2alpha (0, 0.5, 1 and 2 ml/100 ml) were tested and the motility was evaluated immediately after addition of PGF2alpha, after 30 min, 2 h, and 24 h. Two computer-assisted semen analysis (CASA) systems, namely the Sperm Quality Analyzer (SQA-IIC) and the Hamilton Thorne (HTR Ceros 12.1) were used to assess the motility parameters. With the SQA-IIC, sperm motility index values of the treated groups were only slightly higher (P>0.05) compared to the negative control group. The different motility parameters measured with the HTR Ceros 12.1 were similar between the treatment groups, except for beat cross frequency, which was higher in the control group (1.5-5%; P<0.001). This study documented that the addition of 2.5, 5 or 10 mg PGF2alpha to 100 ml diluted boar sperm does not increase any sperm motility parameter. Further research is necessary to elucidate mechanisms by which PGF2alpha in diluted semen may improve the reproductive performance in swine farms.

Animals↗

PubMed2002

Effect of centrifugation on in vitro survival of fresh diluted canine spermatozoa.

Prostatic fluid is unsuitable for preserving dog semen at 4 degrees C and exerts harmful effects upon the spermatozoa during the freezing process. Centrifugation immediately after sperm collection is a common method to remove prostatic admixture. In the present study, dog semen, diluted to 25 x 10(6)/ml, was exposed for 5 min to four different centrifugation speeds (180 x g, 720 x g, 1620 x g and 2880 x g) to determine subsequent sperm losses in the supernatant and to assess sperm survival over time. Using 180 x g as centrifugation speed, 8.9% of the sperm cells was lost upon supematant removal. Using 720 x g, 1620 x g or 2880 x g, sperm losses were lower, 2.3, 0.4 and 0.006%, respectively. After centrifugation, the sperm pellet was rediluted in egg-yolk-Tris extender, cooled and stored for 3 days at 4 degrees C. Motility, progressive motility, membrane integrity and sperm morphology were assessed daily. Acrosomal status was assessed after 3 days of storage. The only functional parameter which was influenced by centrifugation speed was membrane integrity as evaluated by means of SYBR14-PI staining: significantly more dead and moribund sperm cells were found after centrifugation at 1620 x g and 2880 x g after 48 and 72 h of storage at 4 degrees C. When higher initial sperm concentrations (50 x 10(6), 75 x 10(6) or 100 x 10(6)/ml) were evaluated for sperm losses, less than 2.3% of the initial total sperm cells was lost at lower centrifugation speeds. We conclude that centrifuging dog sperm for 5 min at 720 x g is the best strategy to remove prostatic fluid because the loss of sperm cells is acceptable and the functional parameters of the spermatozoa are well preserved, even after 3 days of storage.

Acrosome↗