Biomedical subjects

H Woelders

Publications and source records attributed to H Woelders.

At least 19 recordsLinked to original sources

PubMed2006

Significant variability among bulls in the sperm membrane permeability for water and glycerol: possible implications for semen freezing protocols for individual males.

The aim of this study was to test the hypothesis that bulls have significant intra-individual differences in the hydraulic conductivity (L(p)) and permeability coefficient for glycerol (P(s)) of the sperm cell membrane. The permeability parameters were determined at 22, 10, and 0 degrees C of sperm from 7 Holstein Frisian artificial insemination (AI) bulls, using four ejaculates per bull. A stopped-flow approach was applied to provide temporal resolution sufficient to measure rapid cell volume changes under anisosmotic conditions in the absence or presence of glycerol. This technique utilizes a concentration-dependent self-quenching entrapped fluorophore. The resulting cell volume changes were used in three-parameter fitting calculations to compute L(p) in the absence glycerol, and L(p) in the presence of glycerol (L(p)(gly)) and P(s). Averaged over all bulls, L(p) in the absence of glycerol was 0.28+/-0.01, 0.15+/-0.01 and 0.10+/-0.01 microm min(-1)atm(-1) (mean+/-SD) at 22, 10 and 0 degrees C, respectively, yielding an Arrhenius activation energy (E(a)) of 7.39 kcal/mol. The average L(p)(gly) value at 22 degrees C, was 3.8 times lower than L(p) in the absence of glycerol (P<0.05). L(p)(gly), P(s), and the reflection coefficient (sigma) at 22 degrees C were 0.073+/-0.015 microm min(-1)atm(-1), 0.80+/-0.33 x 10(-3)cm min(-1), and 0.92+/-0.10 (mean+/-SD), respectively. Subsequent experiments were performed at 10 and 0 degrees C. Activation energies for L(p)(gly) and P(s) were 10.08 and 8.77 kcal/mol, respectively. The significant differences between individual bulls in L(p) and P(s) indicate that individual males may require individual adjustments of the cooling protocol. Application of these data in a theoretical model to simulate the osmotic events during freezing resulted in predicted optimal cooling rates in the range of published empirical values.

Analysis of Variance↗

PubMed2006

Animal genetic resources conservation in the Netherlands and Europe: poultry perspective.

Increased global use of highly productive breeds of farm animals has been coupled to loss of genetic diversity in most species. In European countries, various governmental, non-governmental, and private organizations try to preserve genetic diversity of livestock in situ (e.g., by stimulating the use of indigenous, rare breeds by farmers; in nature reserves; or in noncommercial farms). In the case of poultry, maintaining in situ populations of the noncommercial (fancy) breeds largely relies on hobby farmers. In addition to in situ conservation, gene banks are being established for ex situ conservation. In at least 2 countries, France and The Netherlands, there are limited collections of frozen semen of rare poultry breeds. Since 2003, the CGN has started with a more systematic effort to collect, freeze, and store semen of indigenous Dutch poultry breeds. At present, the CGN gene bank contains semen of 11 Dutch rare poultry breeds. Also, CGN has performed research on the methodology for cryopreservation of fowl semen. This recent work was focused on finding a suitable replacement for glycerol, which is contraceptive in the hen, as a cryoprotectant. For reasons of hygiene and sample identification, we favored straw freezing, as opposed to the highly effective pellet freezing method. A significant interaction was found between cooling rate and cryoprotectant concentration. Best post-thaw sperm quality was obtained when combining 0.6 mol of dimethylacetamide/L with a cooling rate of +/- 200 degrees C/min. Inseminations twice per week with 0.3 billion sperm per insemination resulted in 97 and 88% fertilized eggs with fresh and frozen semen, respectively. In 2005, CGN has used this straw freezing method to extend the collection of poultry semen in the Dutch gene bank.

Animals↗

PubMed2005

Improvement of parameters of freezing medium and freezing protocol for bull sperm using two osmotic supports.

The aim of this study was to improve the freezing protocol of bull sperm, by investigating the influence on sperm viability after freeze/thawing of different freezing medium components, as well as the effect of cooling rates in the different stages of the cooling protocol, in single factor experiments. The experimental variables were: (1) salt-based versus a sugar-based medium (Tris versus sucrose); (2) glycerol concentration; (3) detergent (Equex) concentration; (4) presence of bicarbonate; (5) rate of cooling from 22 degrees C to holding temperature (CR1); (6) holding temperature (HT); (7) rate of cooling from holding temperature to -6 degrees C (CR2); (8) rate of cooling from -10 to -100 degrees C (CR3). All experiments were performed using five bulls per experiment (three ejaculates per bull). Sperm motility after freezing and thawing was assessed by CASA system, and sperm membrane integrity was assessed by flow cytometry. Sucrose-based medium did not offer a clear significant benefit compared to Tris medium. The concentration of Equex that gave the best results in Tris-based media group and sucrose-based media group was in a range between 2-7 and 4-7 g/l, respectively. In both media groups, a glycerol concentration of 800 mM was the best in any post-thaw viability parameters. In the Tris media group, the presence of bicarbonate had a negative effect on sperm viability. CR1 and CR2 had no significant effect on any of the post-thaw sperm viability parameters, but a CR1=0.2 degrees C/min and CR2=4 degrees C/min appeared to give better results in both media. The holding temperature (HT) that gave the best results was found to be in the range of 5-9 degrees C. There was a significant disadvantage of using a low CR3 of 10 degrees C/min, while 150 degrees C/min appeared to be the best cooling rate for either medium.

Acrosome↗

PubMed2005

Cryopreservation of boar semen: equilibrium freezing in the cryomicroscope and in straws.

Supercooling causes very abrupt temperature and osmotic changes and can thus lead to freezing damage. Supercooling can be prevented by seeding, using a sample volume and geometry that allows rapid spreading of the ice throughout the sample. In a split-sample comparison of such samples on the cooling stage of a cryomicroscope and seeded at -5 and -15 degrees C, respectively, the percentages of membrane-intact sperm and sperm with acrosomes with a 'normal apical ridge' (NAR) were 72.5+/-3.8 and 75.8+/-2.0 versus 46.3+/-4.8 and 36.0+/-3.7 (means+/-S.E.M., n=4). In ejaculates of 15 unselected AI boars, after seeding at -5 degrees C, the post-thaw % live and % NAR were 66.3+/-10.4 and 74.8+/-7.5, respectively. Our present research is aimed at translating these findings to freezing in straws and at a high sperm concentration. We have designed a novel type of freezing apparatus for controlled-rate freezing of straws, in which supercooling can be effectively prevented in the entire straw. In a split-sample comparison of semen frozen in straws at a sperm concentration of 1.5 x 10(9) cells/ml with nine ejaculates from eight unselected AI boars, we found 54.8+/-1.9% versus 40.7+/-1.7% (means+/-S.E.M.) membrane-intact sperm for the new apparatus and a conventional freezing apparatus, respectively. With bull semen (eight ejaculates from six bulls), we obtained 67.3+/-3.0% versus 59.3+/-2.9% (means+/-S.E.M.) membrane-intact sperm for the new apparatus and conventional freezing, respectively. Additionally, the temperature curve after ice nucleation is of great importance. We have developed a model that allows us to predict that optimal cryopreservation requires a non-linear cooling curve in which the cooling rate varies as a function of subzero temperature.

Animals↗

PubMed2004

Theoretical prediction of 'optimal' freezing programmes.

We have developed a quantitative description of the osmotic behaviour of cells during freezing without a presupposed value of the cooling rate. Instead, at all times the intracellular supercooling is maximised provided that it does not exceed a predetermined value 'p' (e.g., 2 degrees C). This should preclude intracellular ice formation, but also ensures that the osmotic gradient and the CPA concentration gradient are limited, as well as the gradient driven transmembrane fluxes of water and CPA. Using the condition of a constant level of supercooling of p degrees C, equations can be derived to generate non-linear cooling curves in which at all times the cooling rate is maximised (to minimise slow cooling damage), while preventing conditions that could lead to fast cooling damage. Simulations of the osmotic events during freezing, and prediction of the 'optimal' freezing curve can be performed provided that values are available for the membrane permeability coefficients for water (L(p)) and cryoprotectant (P(s)), and their respective activation energies, the initial intracellular osmotically active aqueous volume, and the membrane surface area. Simulations are shown, both with and without permeant solute, to demonstrate how the predicted 'optimal' freezing curve is affected by medium composition, and by membrane permeability and osmotic cell characteristics.

Animals↗

PubMed2004

Capacity of boar spermatozoa to bind zona pellucida proteins in vitro in relation to fertilization rates in vivo.

The purpose of this study was to determine variation among boars in the percentage of sperm in an ejaculate that express enhanced binding of zona pellucida proteins during treatment for capacitation in vitro, and to determine whether this relates to fertilizing ability in vivo. Ejaculates (n=35) were collected from 12 boars. A sample of each ejaculate was treated for capacitation in vitro. During incubation, the zona binding ability of spermatozoa was assessed at regular intervals with fluorescein-conjugated solubilized zona pellucida proteins (FITC-sZP) and propidium iodide, using a flow cytometer. After incubation, a percentage of the sperm had enhanced FITC-sZP binding. The percentage of viable sperm with enhanced FITC-sZP binding, expressed as a percentage of the total sperm population, increased rapidly over the first 60 min and thereafter reached a plateau after 120-180 min. Averaged over all ejaculates, the percentage at 180 min was 46% (range 27-61%); this percentage was significantly different among boars. However, the variation between ejaculates within a boar was relatively small. There was no significant boar effect on the rate at which the percentage of viable cells with enhanced FITC-sZP binding reached the maximum. In ejaculates (n=14) from four boars (selected from the group of 12), we investigated the increase in the percentage of viable sperm with enhanced sZP binding during treatment for capacitation in vitro in relation to the ability to fertilize in vivo. Sows (n=44) were inseminated 4 h after ovulation with a suboptimal insemination dose (0.5x10(9) spermatozoa). Time of ovulation was determined using transrectal ultrasonography and sows were killed at 120 h after ovulation. The percentage of fertilized oocytes, embryo development, and numbers of accessory spermatozoa were determined. The percentage of spermatozoa that were viable and showed enhanced sZP binding after 180 min of incubation was 48 +/- 12% (range 28-56%). The percentage of fertilized oocytes was 85 +/- 27% and 64% of the sows had 100% fertilized oocytes. The percentage of sows with 100% fertilized oocytes correlated well (P< or =0.05, R2=0.98) with the percentage of viable spermatozoa with enhanced FITC-sZP binding after capacitation in vitro.

Animals↗

PubMed2004

Effects of exposure of epididymal boar spermatozoa to seminal plasma on the binding of zona pellucida proteins during in vitro capacitation.

The purpose of the investigation was to determine whether seminal plasma plays a role in the increase during in vitro capacitation of the number of boar spermatozoa with enhanced binding of zona pellucida proteins. Ejaculated spermatozoa and spermatozoa collected from the caudae epididymides of boars were incubated at 39 degrees C in a Tyrode's IVF medium. During incubation, the zona binding ability of individual spermatozoa was assessed with fluorescein-conjugated solubilized zona pellucida proteins (FITC-sZP), using a flow cytometer. Propidium iodide (PI) was included to simultaneously monitor cell viability. During incubation of ejaculated spermatozoa, a percentage of the spermatozoa expressed enhanced binding of FITC-sZP. The percentage of viable spermatozoa with enhanced binding reached a maximum of 37% (S.D.=8, averaged over five boars) after 2-3 h. In epididymal sperm, a similar maximum was observed after incubation in vitro, but a longer time of incubation was needed (6 h). Also, the rate of cell death of epididymal sperm was much lower than that of ejaculated sperm. When epididymal spermatozoa was exposed to seminal plasma in vitro, the time needed to reach a maximal percentage of viable spermatozoa with enhanced FITC-sZP binding was similar to that in ejaculated semen. However, the rate of cell death was still much lower than in ejaculated sperm. We concluded that the binding sites on the sperm surface that are involved in the increased binding of zona proteins during incubation under IVF conditions were not derived from the seminal plasma. The cellular processes leading to the increased binding capacity were accelerated by exposure of the sperm to seminal plasma.

Animals↗

PubMed2004

Determination of bull sperm membrane permeability to water and cryoprotectants using a concentration-dependent self-quenching fluorophore.

The objective of this study was to determine the membrane permeability characteristics of bovine spermatozoa. These included the hydraulic conductivity (Lp), the permeability coefficients (Ps) of four common cryoprotective agents (CPAs) and the associated reflection coefficients (sigma). Stopped-flow fluorometry was applied in order to capture rapid cell volume changes under anisosmotic conditions in the absence or presence of permeant solutes (CPAs). This technique utilizes a concentration-dependent self-quenching entrapped fluorophore. The resulting cell volume changes were used in three-parameter fitting calculations to compute Lp in the absence of permeant solutes and Ps and Lp in the presence of permeating solutes (CPAs) at 22 degrees C. The hydraulic conductivity in the absence of permeating solutes was estimated to be 0.68+/-0.05 microm/min/atm (mean+/-SEM). Hydraulic conductivity (Lp) in the presence of CPAs was 0.91+/-0.27 (mean+/-SEM), 0.29+/-0.04, 0.42+/-0.05, and 0.39+/-0.03 microm/min/atm in the presence of dimethylsulfoxide (Me(2)SO), glycerol, propylene glycol (PG), and ethylene glycol (EG), respectively. The values for Ps were estimated to be 1.72+/-0.36, 1.75+/-0.03, 2.47+/-0.24, and 1.49+/-0.33 x 10(-3)cm/min for Me(2)SO, glycerol, PG, and EG, respectively. The data were used to simulate volume excursions during addition and removal of CPA, to predict the different effects of the four CPAs.

Animals↗

PubMed2003

Neutrophil recruitment and phagocytosis of boar spermatozoa after artificial insemination of sows, and the effects of inseminate volume, sperm dose and specific additives in the extender.

In this study the recruitment of leucocytes and phagocytosis of spermatozoa after artificial insemination of multiparous sows was investigated. In Expt 1, groups of sows received either no inseminate (n = 6) or inseminates with various concentrations of spermatozoa and seminal plasma or different inseminate volumes (n = 9 per group). In Expt 2, groups of sows received inseminates containing no addition, caffeine + CaCl(2), or excess EDTA (n = 6 per group). Leucocytes and spermatozoa were counted in the collected backflow from the vulva, and in the PBS flushings of the genital tract of sows killed at 4 h after insemination. Tissue homogenates were checked for remaining spermatozoa. Leucocyte recruitment did not depend on the presence of seminal plasma or spermatozoa. In the control groups about 43% of the inseminated spermatozoa were found in the backflow and 5% in the genital tract. Many spermatozoa could be recognized inside polymorphonuclear leucocytes. With an inseminate volume of 20 ml instead of 80 ml, fewer spermatozoa were found in the backflow and more (non-phagocytosed) spermatozoa were recovered in the uterus (P < or = 0.05). With a sperm dose of 0.24 x 10(9) instead of 2.4 x 10(9), a higher percentage of the inseminated spermatozoa was recovered in the oviducts (P < or = 0.05). The use of caffeine + CaCl(2) resulted in lower recruitment of leucocytes (P < or = 0.05) and a higher number of non-phagocytosed spermatozoa in the uterus (P < or = 0.01) compared with controls. The numbers of spermatozoa in the oviducts were not different. Insemination with excess EDTA had no positive effects on the number of spermatozoa in the genital tract.

Animals↗

PubMed2003

Autologous and homologous transplantation of bovine spermatogonial stem cells.

The aim of this study was to develop a method for spermatogonial stem cell transplantation into the bovine testis. Five-month-old Holstein-Friesian calves were used and half of the calves were hemicastrated to allow autologous transplantation and the other half were used for homologous transplantation. Approximately 20 g of each testis was used for cell isolation. On average 106 cells per gram of testis containing about 70% type A spermatogonia were isolated. The cells were frozen in liquid nitrogen until transplantation. Testes were irradiated locally with 10-14 Gy of X-rays to deplete endogenous spermatogenesis. At 2 months after irradiation, cells (approximately 10 x 10(6) were injected into the rete testis through a long injection needle (18 gauge), using ultrasonography and an ultrasound contrast solution. At 2.5 months after transplantation, calves were castrated and samples of testes were taken for histological examination. After 2.5 months in the irradiated non-transplanted control testes, only 45% of the tubules contained type A spermatogonia. However, after autologous spermatogonial transplantation, >80% of the tubule cross-sections contained type A spermatogonia. In addition, only 20% of the tubules of the control testes contained spermatocytes and, except for a few tubules (5%) with round spermatids, no more advanced germ cells were found. After autologous spermatogonial transplantation, about 60% of the tubules contained spermatocytes; 30% contained spermatids and in about 15% of tubules spermatozoa were found. No improvement in spermatogonial repopulation was found after homologous transplantation. The results of this study demonstrate, for the first time, successful autologous transplantation of bovine spermatogonial stem cells resulting in a complete regeneration of spermatogenesis.

Animals↗

PubMed2001

Influence of cooling rates and plunging temperatures in an interrupted slow-freezing procedure for semen of the African catfish, Clarias gariepinus.

The objective of this study was to optimize interrupted slow-freezing protocols for African catfish semen. Semen diluted with methanol and extender was frozen in 1-ml vials in a programmable freezer. The temperatures of the freezer (T(chamber)) and of the semen (T(semen)) were measured simultaneously. We first tested two-step freezing protocols with different cooling rates (-2, -5, and -10 degrees C/min) and different temperatures at plunging into liquid N2. The difference between T(semen) and T(chamber) increased with faster cooling rates. In all programs, survival of spermatozoa, expressed as hatching rates, increased from near zero when T(semen) at plunging was higher than -30 degrees C to values equal to those of control when T(semen) at plunging was equal to or lower than -38 degrees C. The inclusion of an isothermal holding period before plunging into liquid N2 (three-step freezing protocols) resulted in an equilibration between T(semen) and T(chamber) and improved semen survival. Semen could be plunged at temperatures as high as -36 degrees C when cooled at -5 or -10 degrees C/min, without compromising postthaw semen survival. Cooling at -2 degrees C/min in combination with a 5-min holding period reduced postthaw survival. We conclude that with slow cooling rates of -2 to -5 degrees C/min, hatching rates can be maximized by plunging as soon as T(semen) reaches -38 degrees C. The isothermal holding period is beneficial when faster rates are used. A simple and efficient protocol for freezing African catfish semen can be obtained by cooling at a rate of -5 to -10 degrees C/min combined with a 5-min holding period in the freezer, at -40 degrees C.

Africa↗

PubMed2001

Phagocytosis of boar spermatozoa in vitro and in vivo.

For successful conception, fertilization-competent spermatozoa must be present at the site of fertilization in adequate numbers until ovulation has taken place. In pigs, a large volume of semen is delivered into the uterus. Most, if not all, of the inseminated liquid is voided from the vulva within a few hours after insemination and approximately 45% of the spermatozoa are lost. Large numbers of spermatozoa are also lost due to phagocytosis by polymorphonuclear leukocytes (PMNs). In pigs, the recruitment of PMNs to the uterine lumen appears to be triggered by insemination of a volume of liquid, rather than by specific components of that liquid or by spermatozoa or seminal plasma. However, persistence of large numbers of PMNs in the uterine lumen at > 12 h after insemination appears to depend on the presence of spermatozoa in the inseminate. In vitro studies have indicated that damaged, killed or capacitated spermatozoa are not phagocytosed preferentially, but that capacitation treatment strongly reduced phagocytosis of spermatozoa. Recent studies have also shown that PMN recruitment and phagocytosis of spermatozoa in vivo can be reduced by addition of caffeine plus CaCl2 to the inseminate, which appeared to have positive consequences for the longer term availability of spermatozoa at the site of fertilization.

Animals↗

PubMed2000

In vitro phagocytosis of boar spermatozoa by neutrophils from peripheral blood of sows.

A considerable number of spermatozoa are used in each sow in routine artificial insemination. However, within a few hours after insemination, many spermatozoa are phagocytosed by polymorphonuclear leucocytes. Some aspects of sperm transport in the female genital tract in the sow have been thoroughly investigated, whereas little is known about the mechanisms involved in the phagocytosis of spermatozoa, or about which spermatozoa (fresh, capacitated or dead) are the most susceptible to ingestion by polymorphonuclear leucocytes. In this study, phagocytosis was investigated by use of an in vitro phagocytosis assay. Polymorphonuclear leucocytes were challenged with either untreated, cold-shocked or frozen-thawed spermatozoa, or with spermatozoa that had been treated to induce capacitation in vitro. The influence of serum on phagocytosis was also investigated. Treatment of the semen to induce capacitation in vitro considerably reduced the phagocytosis of spermatozoa, whereas crude treatments like cold-shock or freezing and thawing reduced phagocytosis only in the first 15-30 min of incubation with polymorphonuclear leucocytes. Viable spermatozoa were phagocytosed mainly through a pathway that was independent of complement or other serum components (for example, antibodies). Complement had little effect on phagocytosis of spermatozoa, but did cause acrosomal exocytosis and cell death.

Acrosome Reaction↗

PubMed1999

Influence of capacitation and fluids from the male and female genital tract on the zona binding ability of bull spermatozoa.

Before fertilization, inseminated spermatozoa acquire the ability to fertilize an egg, a phenomenon called capacitation. Bovine sperm capacitation is influenced by factors originating from both the male and female genital tract, and results in intracellular and membrane changes of the spermatozoa that facilitate the induction of the acrosome reaction. However, the effects of reproductive tract secretions and capacitation on the binding of spermatozoa to the zona pellucida have not been investigated. In this study, a sperm-egg binding assay was used to determine whether the ability of bull spermatozoa to bind to the zona pellucida was altered during in vitro capacitation by heparin or oviductal fluid, or by treatment of spermatozoa from the cauda epididymidis with accessory sex gland fluid. In addition, biotinylated solubilized zona pellucida proteins were used to visualize zona binding on spermatozoa. The ability of bull spermatozoa to bind to the zona pellucida was increased after both heparin and oviductal fluid induced in vitro capacitation. Exposure of spermatozoa from the cauda epididymidis to accessory sex gland fluid resulted in a direct increase in zona binding ability, followed by a further increase during capacitation in vitro. Binding of solubilized zona proteins was restricted to the acrosomal cap of bull spermatozoa. It is suggested that the observed increased ability of bull spermatozoa to bind to the zona pellucida enables optimal sperm-egg attachment, which also relates to the induction of the acrosome reaction by the zona pellucida. Thus, increased zona binding ability is likely to be an essential part of the process of capacitation.

Animals↗

PubMed1998

Enhanced binding of zona pellucida proteins to the acrosomal region of the intact boar spermatozoa in response to fertilizing conditions: a flow cytometric study.

In this investigation we sought to determine whether sperm capacitation in vitro is accompanied by changes in the functional presence of zona binding sites on the plasma membrane of boar spermatozoa. During sperm incubation at 39 degrees C in various modifications of a Tyrode's-based in vitro fertilization medium, the zona binding ability of individual spermatozoa was assessed with fluorescein-conjugated solubilized zona pellucida proteins, using a flow cytometer. Propidium iodide was routinely included to allow simultaneous assessment of membrane integrity; rhodamine-conjugated peanut agglutinin was used to assess acrosomal status. During incubation in the fertilization medium, a subpopulation of live acrosome-intact spermatozoa developed enhanced binding of the fluorescein-conjugated solubilized zona proteins. Microscopy revealed that the increase in cytometrically detected zona binding was paralleled by an increase in the area on the sperm head to which zona proteins bound, from the apical region to the whole of the acrosomal region. The changes were accelerated by phosphodiesterase inhibitors, were attenuated by omission of bicarbonate, and were completely inhibited by addition of EGTA. In the fertilization medium, numbers of sperm showing enhanced zona binding maximized after 60-90 min. This time course is somewhat similar to that reported by others for development of egg-penetrating ability in vitro. We suggest that the observed changes in zona binding ability bring about optimal sperm-egg attachment; they may also relate to induction of the acrosome reaction by zona pellucida components. In consequence, the zona binding changes may be an important part of the process by which the sperm acquires fertilizing ability as a result of capacitation.

Acrosin↗

PubMed1997

Identification of bovine zona pellucida glycoproteins.

Despite the economical importance of in vitro gamete technologies in cattle, only little is known about the molecular mechanisms of binding of spermatozoa to the zona pellucida (ZP) of the oocyte. The aim of the present work was to identify proteins from the bovine zona pellucida (bZP) and to investigate which bZP proteins play a role in sperm-egg binding. High resolution 2-dimensional polyacrylamide gel electrophoresis of bZP proteins under reducing conditions showed that the bovine ZP could be separated into 4 glycoprotein spots, provisionally named bZP1, bZP2, bZP3, and bZP4, with different molecular masses and isoelectrical points. The N-terminal amino acid sequence of bZP1, bZP2, and bZP4 could be determined. The N-terminal amino acid sequences of bZP1 and bZP4 were identical and were homologous to that of pZP4. Comparison of our data to that of Noguchi et al., 1994 (Biochim Biophys Acta 1201:7-14) revealed that bZP2 and bZP4 are fragments of bZP1. Immunoblot analysis showed that, respectively, anti-porcine-ZP3alpha and -ZP3beta antibodies recognized 2 distinct regions of the bZP3 spot. Both antibodies inhibited sperm-egg binding in the bovine. We conclude that the bovine ZP consists of 3 proteins that correspond by size, N-terminal amino acid sequence, and antigenic determinants of pZP1, pZP3alpha, and pZP3beta, respectively, that are encoded by the porcine ZPA, ZPB, and ZPC genes (Harris et al., 1994: J Seq Map 4:6331-393), respectively.

Animals↗

PubMed1997

Effects of trehalose and sucrose, osmolality of the freezing medium, and cooling rate on viability and intactness of bull sperm after freezing and thawing.

The influence of the presence of trehalose or sucrose, the osmolality of the freezing medium, and the cooling rate on success of cryopreservation of bull sperm were investigated. Bull semen was frozen at four different cooling rates varying from 40 to 300 degrees C/min in standard Tris-egg yolk medium and in hypertonic or isotonic media containing 0.2 M trehalose or sucrose. Sperm motility, H33258 exclusion, and acrosome morphology were evaluated. In the hypertonic media the motility was almost completely inhibited, but could partly be restored by returning to isotonic conditions. Medium composition and cooling rate had significant effects, with a strong interaction of medium x cooling rate. The optimal cooling rate was found to be between 76 and 140 degrees C/min. At the highest cooling rate, both the presence of the sugars and the high osmolality of the hypertonic media conferred significant protection against fast-cooling damage, sucrose being significantly better than trehalose. At all other cooling rates the medium differences were less pronounced, but the isotonic sugar media were generally superior to standard medium as to preservation of acrosome intactness and H33258 exclusion. This indicates that the isotonic sugar media could enable some improvement of cryopreservation of bull semen over the currently used Tris-egg yolk medium. Further improvement of sperm survival at the AI station should be possible by implementation of controlled rate freezing, using a cooling rate in the optimal range between 76 and 140 degrees C/min.

Animals↗

PubMed1997

Fundamentals and recent development in cryopreservation of bull and boar semen.

This paper describes some of the effects of cryopreservation on living sperm, and some recent results for bull and boar semen. During freezing and thawing of semen, the sperm can be damaged by the rapid and dramatic changes in the physicochemical conditions during cooling and ice formation. The rate and extent of these changes, and their impact on the cells, are strongly affected by the cooling rate and the medium composition. For many species, including humans and several farm animals, the optimal cooling rate has been determined, sometimes in combination with the cryoprotectant concentration. Recent results indicate that the cryopreservation of bull semen at the AI station could be improved by using controlled freezing at a cooling rate of about 100 degrees C/min, and by changing the composition of the freezing medium. An interaction between glycerol concentration and cooling rate has been described for boar semen. Current cryopreservation methods for boar semen based on an optimal combinations of these two factors allow consistent sperm survival in the frozen semen of AI boars, with acceptable variation among boars. The fertility results for semen processed in this fashion seem to be adequate for special applications, such as export, or long-term storage.

Animals↗