Number Of Viable Spermatozoa Research Articles

Cushioned and high-speed centrifugation improve sperm recovery rate but affect the quality of fresh and cryopreserved feline spermatozoa

The development of endoscopic transcervical catheterization (ETC) in the queen increases the interest in handling fresh and cryopreserved feline semen. The ETC requires a small volume of the insemination dose with a high concentration, not easily reached with the actual frozen technique in this species. Centrifugation is widely used to concentrate spermatozoa for several purposes, but this process is detrimental to spermatozoa. This study verified the effects of conventional and cushioned centrifugation on fresh and cryopreserved feline spermatozoa. To this, semen was collected from 20 toms, grouped in seven pools and diluted. After dilution, the pools were divided into two aliquots, the first used for centrifugation on fresh semen, and the second, after freezing, on cryopreserved semen. Centrifugation regimens were: conventional at 500×g, conventional at 1000×g, and cushioned (iodixanol) at 1000×g. The sperm recovery rate was calculated for the three centrifugation regimens, and sperm kinematics, membrane and acrosome integrity, and plasma membrane stability on viable spermatozoa were assessed as endpoints.The data reported in this study showed that the centrifugation at 500×g resulted in negligible effects on both fresh and cryopreserved spermatozoa, but the lower recovery rate (62.4 ± 3.1 % and 60.2 ± 1.6 %, respectively) underlines the loss of a large proportion of spermatozoa, unfavourable in a species with small total sperm ejaculated. On the other hand, the centrifugation at 1000×g improved the recovery rate (86.9 ± 4.3 % and 89.8 ± 2.4 % in fresh and cryopreserved samples, respectively), but was more deleterious for feline spermatozoa, especially in cryopreserved samples (i.e. total motility of 40.7 ± 5.4 % compared with 57.2 ± 9.8 % in cryopreserved uncentrifuged samples, P < 0.05), resulting in artificial insemination doses of lower quality. The recovery rate in cushioned centrifugation appeared less efficient, likely due to the small volume of feline samples, which makes difficult the separation of sperm pellet and cushioned fluid. Interestingly, in cryopreserved samples centrifuged at 1000×g the number of viable spermatozoa with membrane destabilization (31.3 ± 3.2 %) was greater than uncentrifuged (4.1 ± 0.7 %, P < 0.05) and those centrifuged at 500×g (9.8 ± 1.3 %, P < 0.05), suggesting modifications induced by the cryopreservation amplifies centrifugation sublethal damage on feline spermatozoa. Cushioned centrifugation on cryopreserved samples showed kinematics similar to uncentrifuged samples, but higher viable spermatozoa with membrane destabilization (37.4 ± 3.4 % vs 4.1 ± 0.7 %; P < 0.05). In felines, g-force is crucial for sperm recovery rate during centrifugation, with better results at 1000×g; on the other hand, greater g-forces could have a significant impact on the quality of feline insemination dose, especially in cryopreserved samples.

Cryopreservation of Sperm: A Review.

Sperm cryopreservation has been used as a sperm preservation solution for infertility issues faced by men undergoing cancer treatment for over 40 years. Recent developments in sperm cryopreservation and its wide variety of therapeutic uses are discussed in this article, which offers a succinct and up-to-date overview of the relevant literature. Recently, sperm cryopreservation has been employed for a wider variety of therapeutic purposes. As a result, sperm freezing is becoming available to a wider variety of patients, which requires more specialized personnel and increases overhead expenses. While sperm cryopreservation before cancer treatment is accessible in many countries, oncology doctors' referral rates and patient participation with cryopreservation services have been observed to be poor. In addition, there are still moral concerns with sperm banking, including whether or not donors' identities should be protected and whether or not a deceased person's sperm should be used after his or her death. This article discusses the recent developments in sperm cryopreservation technology and the moral questions that have arisen around this practice, with an eye toward how a deeper knowledge of these concerns can help more people get access to treatments that might help preserve their fertility. A sperm bank will notify clients about the screenings it does and the background information it gathers on individual donors to guarantee the safety and quality of the sperm they get. The viability and quantity of viable sperm in a thawed sample are often guaranteed by a sperm bank. They will look for very fertile males who can donate sperm that can withstand the freezing and thawing procedure. In many cases, sperm banks advertise their samples as having a certain number of viable sperm per milliliter, and they may provide many sample types for various applications (intracytoplasmic sperm injectionand intrauterine insemination).

Seasonal Variability in Cryoresistance of Saanen Goats Spermatozoa and Reproductive Characteristics

Cryopreservation of gametes is one of the important stages of assisted reproductive technologies (ART). To obtain high results using ART, it is essential to cryopreserve sperm in a favorable season, when gametes have a high ability to fertilize oocytes. The aim of this research was to study seasonal variability of spermatozoa cryoresistance and reproductive characteristics of Saanen goats. After removing the seminal fluid, the gametes of five mature animals were cryopreserved in a solution of 10% glycerol and 20% egg yolk. The ability of spermatozoa to fertilize was examined by the embryo development rate on day 7 of in vitro culture. Reproductive characteristics of Saanen goats during the breeding season were found to be featured by increased sperm concentration and motility. The survival rate of goat spermatozoa after cryopreservation is seasonally dependent, i. e. in the breeding season the number of viable spermatozoa was notably higher than in non-breeding one. The frequency of oocyte fertilization after the use of fresh and cryopreserved spermatozoa derived in different seasons was significantly higher in the breeding season group. The number of embryos which developed to the blastocyst stage was the lowest after fertilization of oocytes with cryopreserved spermatozoa obtained in the non-breeding season. It is concluded that sperm cryotolerance and reproductive characteristics of goats are higher in a breeding season compared to non-breeding one. The findings will allow to develop a strategy in the reproduction of farm animals for the food security of Ukraine.

Reproductive Aging of African Catfish in Aquaculture

The work is devoted to the study of reproductive aging of African catfish in the conditions of industrial aquaculture. The problem is urgent, because industrial aquaculture changes the biology of African catfish so much that it loses its ability to reproduce naturally. The offspring of African catfish can be obtained only with the use of hormonal inducers of gametogenesis. Questions of age selection of producers and age composition of breeding stock in this type of fish are still open. In practice, we have to face the facts of poor quality of sexual products in primary spawning or old females and males. The aim of the study was a comparative assessment of age-related variability of reproductive properties of female and male African catfish in the conditions of industrial aquaculture. The results of our study showed the presence of age-related dynamics in the quality of sexual products in African catfish. Age-dependent differences in the quality and fertilization of eggs, the viability of embryos and larvae, their size, and the quality of offspring were established. When studying the properties of sexual products in fish of different ages, differences in morphometric and physiological parameters were found for a complex of indicators such as the size and diameter of eggs, sperm concentration, and the number of viable spermatozoa. Evaluation of the influence of parents age on the viability of offspring in the embryonic and postembryonic periods revealed that this indicator is the lowest in first – spawning fish, and the highest in middle-aged fish. The Russian Foundation for Basic Research has supported our study with the grant No. 18-416-730005.

Causes and Impact of Cryopreservation-Associated Damage on Different Parameters of Human Spermatozoa and its Clinical Impact

Sperm cryopreservation has been widely used for assisted reproductive technology (ART). Indications for sperm cryopreservation include donor insemination, cryopreservation prior to surgical infertility treatment, and malignancies to avoid additional surgery in couples undergoing repeated in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI) cycles. However, dramatic changes during cryopreservation have detrimental effects on the sperm membrane, resulting in a large increase in the percentage of poorly motile sperm or sperm with abnormal morphology. The negative effects related to rapid temperature decrease, such as osmotic injury, cellular dehydration, intracellular ice crystal formation, and oxidative stress can also damage the sperm in ways that affect reproductive outcome. This comprehensive review focusses on describing the detrimental effects of the cryopreservation process on sperm and aims to clarify that not all impaired sperm parameters have the same impact on the clinical practice of ART. Regarding the parameters studied, some of the biomarkers used for sperm maturity, hyaluronic acid binding capacity, or damaged DNA have limited clinical significance compared to other semen parameters which provide more useful information for clinical practice and are often dismissed, such as total motility or total motile sperm count (TMSC). In the authors’ experience, TMSC gives valuable quantitative information about the number of viable spermatozoa. Indeed, TMSC should be assessed specifically for groups of patients in which sample availability is limited.

Reproductive Senescence in Drones of the Honey Bee (Apis mellifera).

In the face of high proportions of yearly colony losses, queen health and fecundity has been a major focus of industry and research. Much of the reproductive quality of the queen, though, is a function of the mating success and quality of the drones (males). Many environmental factors can negatively impact drone semen quality, but little is known about factors that impact the drones’ ability to successfully mate and deliver that semen, or how widely drones vary. In our study, we observed the daily variation in honey bee drone reproductive quality over time, along with a number of morphological traits. Drones were reared in cages in bank colonies, and 20 individuals were dissected and measured daily. The number of viable spermatozoa in the seminal vesicles was zero at emergence and reached an average maximum of 7.39 ± 0.19 million around 20 days of life. Decline in spermatozoa count occurred after day 30, though viability was constant throughout life, when controlling for count. Older drones had smaller wet weights, head widths, and wing lengths. We predict that this is likely due to sampling bias due to a differential lifespan among larger, more reproductively developed drones. Our study shows that drones are more highly variable than previously suggested and that they have a significant variation in reproductive physiology as a function of age.

Relationship between pre-pubertal biometrical measures and sperm parameters for the selection of high genetic merit pure and crossbred boars

The determination of specific reproductive parameters, which allow the early selection of high genetic merit boars from different lines, is critical as it may avoid the high maintenance costs of keeping unfertile males in the production system. The aim of this work was to evaluate body and testicular measurements during the pre-pubertal phase, and associate them with reproductive characteristics, such as precocity and libido, and seminal characteristics during puberty in two different lines. Two pure breeds used in the crossing of female lines (FL) and two crossbred terminal lines (TL) were evaluated through body and testicular biometrical measures and seminal characteristics. Terminal line boars presented greater body weights and testicular measures (P < 0.01) compared to FL animals throughout the pre-pubertal period. TL males had their first collection at 24.0 ± 0.3 weeks, while their FL counterparts started about one week later (25.0 ± 0.3 weeks, P < 0.05) and age of selection presented a two-week delay in FL males (29.0 ± 0.7 versus 27.0 ± 0.6 weeks). Overall, 57% of FL boars were selected up to 31 weeks of age, while 90% of TL males were selected during the same period (P < 0.05). It was observed that genotype did not affect the seminal characteristics evaluated: volume, concentration, number of total spermatozoa, number of viable spermatozoa or sperm motility and kinetics. However, TL crossbred males showed higher percentage of normal spermatozoa and lower percentage of tail and head defects (P < 0.05). Sperm concentration in the 28th week was positively correlated with body weight in the 1st (r = 0.58, P < 0.001) and 15th (r = 0.39, P < 0.05) weeks of age. Moreover, sperm concentration in the 28th week was also correlated with right testicle length (RTL) in the 3rd week (r = 0.40, P < 0.05), and right testicle width (RTW) at week 15 (r = 0.36, P < 0.05). There was a negative correlation between RTW at week 15 and age of selection (r = −0.32, P < 0.05). Therefore, body and testicular measurements in the pre-pubertal period may predict semen concentration and can be used as early classification criteria for the selection of boars from different lines, without the risk of culling high value boars.

Fluorescent multiple staining and CASA system to assess boar sperm viability and membranes integrity in short and long-term extenders

The aim of this study was to assess the effect on boar spermatozoa quality of in vitro storage in short and long-term extenders by fluorescent multiple staining (FMS) and computer assisted semen analyzer (CASA). Fresh ejaculates from three healthy, sexually mature boars were diluted with equal volumes of six short-term or three long-term commercial extenders and stored at 19°C for 6 days (short-term) or 12 days (long-term). The integrity of spermatozoa membranes was analyzed by FMS using propidium iodide, 5,5’,6,6’-tetrachloro-1,1’,3,3’ tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) and fluorescein isothiocyanate-conjugated peanut agglutinin (PNA). The results obtained from this staining were compared with spermatozoa motility assessed by CASA. Our study showed that the number of viable spermatozoa with non-reacted acrosomes and intact mitochondria was positively correlated with the rate of motile spermatozoa (r2&gt;0.9) irrespective of the extender used. In all extenders the number of motile spermatozoa significantly decreased as preservation period increased (P&lt;0.05). FMS test is a potent indicator of sperm motility because it analyses mitochondrial integrity independently from observable alterations in motility. The best performing extenders were BTS for short-term storage and TRI-x-Cell for long-term storage.

CORRELAÇÃO DA MORFOLOGIA ESPERMÁTICA EM PROGRAMAS DE INSEMINAÇÃO ARTIFICIAL CONVENCIONAL EM NOVILHAS E VACAS DA RAÇA NELORE

The number of doses per pregnancy gives an indication of the fertility of the cow, the quality of semen and insemination. Therefore, the goal of this study was to investigate the relationship between the physical and morphological assessment of semen cryopreserved in Central with the number of doses per pregnancy and pregnancy rate by the number of doses used. Four Nelore bulls were challenged in 557 heifers and one bull in 156 Red Angus cows, both Nellore. For number of insemination doses required per pregnancy, tended to correlate significantly with lower amount of pathologies (0.053, P&gt; 0.05), with no significant correlation between sperm motility (0.433, P&gt; 0.05), spermatic vigor (0.932 , P&gt; 0.05), concentration (0.111, P&gt; 0.05), number of viable sperm (0.637, P&gt; 0.05), percentage of major diseases (1, P&gt; 0.05) and percentage of pathologies Total (0.623, P&gt; 0.05). For pregnancy rate, no significant correlation with sperm motility (0.218, P&gt; 0.05), spermatic vigor (0.252, P&gt; 0.05), sperm concentration (1, P&gt; 0.05), number of viable sperm (0.637, P&gt; 0.05), percentage of major diseases (0.395, P&gt; 0.05), percentage of pathologies smaller (0.740, P&gt; 0.05) and percentage of total conditions (0.872, P&gt; 0.05 ). For other parameters, there was a significant effect of sperm motility and (0.041, P &lt;0.05), and sperm motility and number of viable sperm (0.042, P &lt;0.05). The conclusion of this study that there was no significant correlation between the andrological examinationof post-thaw semen, number of insemination doses needed for pregnancy and pregnancy rate by the number of doses, with only significant correlation between sperm motility and vigor, and sperm motility and percentage of viable sperm

The effect of swim-up purification and incubation of cells on sperm viability in dogs of different ages

The influence of selected semen extenders on the motility of frozen-thawed dog spermatozoa has been clearly demonstrated in several studies, although there are no reports indicating the effect of swim-up purification on sperm viability in this species of mammals. Therefore, this study was aimed at investigating phosphatidylserine (PS) externalization and necrosis in sperm after variable lengths of time of in vitro incubation after swim-up purification. Dog semen samples were collected from (i) ten dogs aged six months to 1.5 year, (ii) ten dogs aged six to eight years, and (iii) ten dogs aged 11 to 13 years. A flow cytometric method was employed to evaluate dog sperm viability in animals of different age groups after employment of a swim-up (SU) purification technique. After SU spermatozoa were incubated for 15, 30, 45 and 60 min in Sperm-TALP medium. We observed an increase in the number of viable sperm (double negatives) after 15 min of incubation compared to sperm undergoing PS externalization and late necrotic sperms (P &amp;lt; 0.001) in each group of dogs. We also found a higher number of early necrotic sperm after 60 min of in vitro incubation (P &amp;lt; 0.001). The amounts of late necrotic sperm and cells with PS externalization were similar among animals of different age groups. We show for the first time that most viable sperm are recovered after an in vitro incubation step of 15 min (control samples in this study) because as the time of incubation increases so does the number of degenerated or damaged cells. The higher number of early necrotic cells after 60 min of in vitro incubation may be a special feature of this species and may result from the induction of necrosis in the sperm. This knowledge may be used in future experiments for the preparation of spermatozoa following in vitro fertilization in dogs.

Copper Metabolism Disorders Affect Testes Structure and Gamete Quality in Male Mice

In the present study, animals with a genetic defect in copper metabolism were used as a model organism to study the role of copper in reproduction and to determine whether the disturbances in copper and zinc metabolism affect the testicular tissue and gamete quality in males. Mice with an X-linked mosaic mutation (Atp7amo-ms) exhibit pathological features characteristic of affected copper metabolism. This mutation usually leads to lethality of the mutant males which generally expire on about day 16. Only 4% of mutant animals survive the critical period, achieve maturity, and become fertile. To improve the mutants' viability they were treated with subcutaneous injections of cupric chloride. We measured copper and zinc concentration in the gonads of young (14-day-old) and adult (5-month-old) mutant and control males. Results indicate that copper content was increased but zinc was decreased in the mutant testes. Analysis of the morphology of the testis of the young animals indicate that apoptosis (characteristic for the gonads of young males) was increased in the gonads of the 14-day-old mutants. This process was less advanced in the group of 14-day-old copper treated control males. Apoptosis was also increased in the testes of the adult mutants. Moreover in adult mutants we observed pathological changes in testes morphology (atrophic and sclerotic tubules). Copper and zinc disorders also negatively influenced semen quality parameters, including sperm motility, head morphology, tail cytoplasmic membrane integrity, and number of viable spermatozoa. Poor semen quality of the mutant males seems to be responsible for affected in vivo fertilization efficiency. Treatment with cupric chloride did not influence semen quality except in maturation rate, which was even slower in both mutant and control males after treatment. Additionally, in mutants, copulatory plugs and fertile copulation outcome were decreased after copper treatment.

Within-population variation in ejaculate characteristics in a prolonged breeder, Peron’s tree frog, Litoria peronii

Sperm number is often a good predictor of success in sperm competition; however, it has become increasingly clear that, for some species, variation in probability of paternity cannot be explained by sperm number alone. Intraspecific variation in ejaculate characteristics, such as the number of viable sperm and sperm longevity, may play an equally important role in determining fertilization success. Here, we assess variation among ejaculates in three factors that may contribute to fertilization success (number of sperm per ejaculate, viability, and longevity), in a population of Peron's tree frog (Litoria peronii). We detected large variation among males in the number of sperm per ejaculate and the proportion of viable sperm within ejaculates, which could not be explained by variation in either male size or body condition. However, the proportion of viable sperm released by males increased over the season. Finally, we assessed sperm longevity (proportion viable sperm determined using a dual-fluorochrome vital dye) at two different temperatures. At 23 degrees C, on average, 75% of sperm remained viable after 2 h, but there were significant differences amongst males with the percentage of viable sperm ranging from 43% to 95%. For sperm incubated at 4 degrees C, ejaculates varied fivefold in sperm longevity with some males having 50% viable sperm after 5 days. Our data suggest that ejaculate characteristics (sperm number, viability, and longevity) vary widely in Peron's tree frog and may therefore play an important role in determining siring success both in the presence and absence of sperm competition. We discuss the results in relation to selection on ejaculate traits via natural and sexual selection in this and other amphibians.

263 EVALUATION OFALTERNATIVE CRYOPROTECTANTS AND CHOLESTEROL-LOADED CYCLODEXTRINS FOR INCREASING CRYOSURVIVAL OF SEXED REFROZEN STALLION SPERMATOZOA

Commercial use of sex-sorted stallion sperm will depend upon the ability to cryopreserve the sperm. In many cases the desired sperm for sorting is already cryopreserved; therefore, this will necessitate the need to thaw, sex-sort, and subsequently refreeze the sperm for future use in intracytoplasmic sperm injection (ICSI). Experiments were conducted to determine if previously frozen stallion spermcould be thawed, sex-sorted, and cryopreserved. Ejaculates from 6 stallions were frozen in FR5 cryopreservation medium at 400 million sperm mL–1 (0.5-mL straws). Samples were thawed for 30 s in 37�C water and diluted (100 million sperm mL–1) in Kenny's modified Tyrode's medium (KMT). Samples (2 mL) were stained with 36.53 µm Hoechst 33342 in 34�C water for 45 min and then diluted to 75 million sperm mL–1 with KMT containing 0.002% food coloring dye (FD&amp;C #40). Sperm were sorted using an SX MoFlo™ (Dako, Fort Collins, CO, USA), and the sorted samples centrifuged at 850g for 10 min. The sperm were suspended to 4.6 million sperm mL–1 in FR5 + 0.54 m glycerol (control) or the following treatments: FR5 + 0.52 m dimethylformamide (DMF); FR5 + 0.54 m glycerol + 1.5 mg cholesterol-loaded cyclodextrins (CLC)/120 million cells; FR5 + 0.52 m DMF + 1.5 mg CLC/120 million cells. Non-sorted control sperm (NSC) were removed from original frozen/thawed samples and suspended to 4.6 million sperm mL–1 in FR5 + 0.52 m DMF. Sorted and non-sorted samples were cooled from 22�C to 4�C over 2 h, loaded into 0.25-mL straws, and frozen in liquid nitrogen vapor. Straws were thawed in 37�C water for 30 s and the percentages of motile and viable sperm were determined using computer-assisted sperm analysis and flow cytometry (5 million cells were stained with 10 µL propidium iodide and 20 µL SYBR-14), respectively. Data were analyzed by analysis of variance and treatment means were separated using the Student-Newman-Keuls mean separation test. The percentages of total motile spermatozoa were higher for frozen/thawed sexed refrozen (FTR) sperm refrozen in DMF + CLC (10%) than for the glycerol control (2%; SEM � 1; P &lt; 0.05), but this motility was similar to that of the DMF control (5%), the glycerol + CLC treatment (4%), and the NSC (7%; SEM � 1; P &gt; 0.05). The percentages of viable spermatozoawere higher for FTR (glycerol and DMF) sperm treated with CLC prior to freezing (68% and 71%) than for control sperm (58% and 56%; SEM � 3; P &lt; 0.05), respectively. Frozen/thawed sexed refrozen samples resulted in significantly higher percentages of viable spermatozoa compared to NSC (42%; SEM � 3; P &lt; 0.05). These findings indicate that cryopreserved stallion sperm can be thawed, sex-sorted, and refrozen and still maintain a high percentage of viable spermatozoa; treating sperm with CLC prior to refreezing improved cryosurvival for both cryoprotectants. This may provide adequate numbers of viable sperm for use in intracytoplasmic sperm injection procedures. This work was funded by XY, Inc.

Egg Fertility in Domestic Fowl Gallus gallus domesticus as a Function of the Yolk Surface Area

The increase of egg mass and reliable decrease of egg fertility were observed when the mass surface area of yolk (egg) increased. The results obtained suggest that, in addition to the number of viable spermatozoa penetrating across the perivitelline membrane within 15–20 min after ovulation, the probability of fertilization depends on the area of egg surface, which approximately corresponds to the area of perivitelline membrane. Apparently, the ratio of receptors′ numbers and spermatozoa, which contact with them on the germ disc surface, to their number on the non-germ zone decreases with the increase of yolk size. The fertility depression associated with the increased area of perivitelline membrane suggests that the egg yolk size is one of the factors of fertility of the female gametes in terms of both egg content variability and aging.

Differences among breeds and manifestation of heterosis in AI boar sperm output

A total of 271,547 records of semen collections were utilized to appraise sperm characteristics of 3319 boars belonging to eight breeds: Czech Large White (CLW), Czech Landrace (CLA), Přeštice Black-Pied (PBP), Czech Meat Pig (CM), Hampshire (HA), Duroc (DC), Pietrain (PN), Large White (LW), and various crosses of these breeds. The data was collected over 8 years (1990–1997) from insemination stations for boars in the Czech Republic. The assessment of sperm output was based on semen volume, number of total spermatozoa and number of viable spermatozoa. A linear model was used for statistical analysis included fixed effects of breed or crossbred combinations, boar within breed or crossbred combinations, year–season, and linear and quadratic regression on age of boars at collection and on interval between collections. The average semen volume of boars ranged from 161 to 349 ml, number of total spermatozoa from 81×10 9 to 119×10 9 and number of viable spermatozoa from 60×10 9 to 86×10 9. The lowest values were detected in DC while the highest were observed in LW. In general, sperm output significantly differed across breeds and their crossbreeds. The highest heterosis effect for semen volume was 30.6% (HA × PN), for number of total spermatozoa 18.2% (HA × PN) and 10.4% for number of viable spermatozoa (CLA × DC). Sperm output varied with season, including high values in autumn and winter and low ones in spring and summer.

The Effect of Antifreeze Glycoproteins on Survival of Fish Spermatozoa under the Conditions of Long-Term Storage at 4°C

It was already shown that antifreeze glycoproteins isolated from the blood of fish occurring in circumpolar regions inhibited the growth of ice crystals both in vitro and in vivo. When the spermatozoa were frozen to liquid nitrogen temperature, addition of antifreeze glycoproteins to cryoconserving media made it possible to decrease twofold the concentration of the synthetic cryoprotector dimethylsulfoxide without the loss and even with a certain increase in the number of viable spermatozoa. This effect was observed in the case of combined, rather than separate, addition of the fraction of weakly active (low molecular weight) and active (high molecular weight) antifreeze glycoproteins. Here, we studied the effect of antifreeze glycoproteins on the survival of carp spermatozoa under storage at 4°C for varying periods of time. In the presence of total fraction of low and high molecular weight antifreeze glycoproteins (2 and 10 mg/ml) added in a physiological proportion (3 : 1), the survival of spermatozoa increase but this increase did not depend linearly on the medium concentration of protein. Low and high molecular weight antifreeze glycoproteins added separately (10 mg/ml) either did not affect or slightly affected the preservation of cells. The hypothermic effect of antifreeze glycoproteins in water was significantly higher than in a medium with salt activator.

Assessment of sperm characteristics post-thaw and response to calcium ionophore in relation to fertility in Swedish dairy AI bulls

The present study examined the relationship between bull sperm characteristics post-thawing, after swim-up, and after challenge to calcium ionophore in relation to fertility (56-d nonreturn rates) after artificial insemination (AI). Spermatozoa from 25 semen batches derived from 15 Swedish Red and White AI bulls were evaluated with regard to post-thaw motility, membrane integrity, and migration through a swim-up procedure. The swim-up separated spermatozoa were assessed in terms of sperm concentration, viability and capacitation status as well as their response to exogenous calcium ionophore (A23187). Acrosome reactions were evaluated by fluorescence microscopy and flow cytometry. Sperm motility and viability post-thawing were significantly correlated with fertility. For the swim-up separated semen, significant correlations to nonreturn rates were found for concentration, viability, number of viable spermatozoa and sperm capacitation status (Pattern F and Pattern B). The only parameter significantly correlated to fertility after the ionophore challenge was the percentage of acrosome-reacted spermatozoa with remaining equatorial fluorescence, as assessed by fluorescence microscopy, but not by flow cytometry. The regression analysis showed that combining the results of sperm membrane integrity assessment post-thawing with those of capacitation status after swim-up provided the best prediction of fertility. The accuracy of prediction did not improve when these parameters were combined with the percentage of spermatozoa in which acrosome reaction was induced by ionophore challenge.

Absence of colony-stimulating factor-1 in osteopetrotic (csfmop/csfmop) mice results in male fertility defects.

Previous studies have shown that the mononuclear phagocyte growth factor, colony-stimulating factor-1 (CSF-1), has an important role in female reproduction. Mating experiments with osteopetrotic (csfmop/csfmop) mice, which possess an inactivating mutation in the CSF-1 gene, suggested that there are male, as well as female, reproductive defects. In the present study, we have shown that male csfmop/csfmop mice have a sevenfold lower concentration of circulating testosterone (T) and a significantly lower intratesticular T concentration than wild-type mice. These lowered T concentrations were associated with a reduction in mating capability and a reduction in the number of viable sperm. Reconstitution of male csfmop/csfmop mice with either circulating T in the adult or circulating CSF-1 throughout the postnatal period completely restored viable sperm numbers and significantly restored sexual behavior. These observations, coupled with the close association of Leydig cells with testicular macrophages and the proposed function of these macrophages in the regulation of Leydig cell steroidogenesis, suggest that CSF-1-regulated testicular macrophages play an important role in male reproduction.

A note on conception rates and litter sizes following the intrauterine insemination of ewes at an induced oestrus during seasonal anoestrus

Priming with a progestagen-impregnated vaginal pessary followed by an injection of 750 i.u. pregnant mare serum gonadotropin at pessary withdrawal was used to induce oestrus and ovulation during anoestrus (13 June, latitude 57°N) in 58 Border Leicester × Scottish Blackface ewes that had lambed previously in late March/early April. At 54 to 58 h after pessary withdrawal the uterus of each ewe was viewed by laparoscopy and 50 × 106 viable sperm deposited into each uterine horn. Mean ovulation rate was 2·9 (s.d. 0·12; range 1 to 5). Plasma progesterone concentrations remained &gt;1·5 μg/l in 54 (93%) of the ewes until at least day 21 after insemination implying a high fertilization rate. After allowing for three ewes that were observed to abort between 39 and 55 days after insemination and another three, carrying nine normally developed lambs, that died between 102 and 136 days of gestation, 40 of the remaining 48 ewes (i.e. 69% of the original 58) produced 76 viable lambs in early November. These conception and lambing rates obtained following the intrauterine insemination of 100× 106 viable sperm per ewe are more than double those obtained following natural mating or conventional cervical insemination with four times the number of viable sperm and imply that intrauterine insemination may enhance lamb production from ewes induced to breed during seasonal anoestrus.

Current role of semen storage and artificial insemination in the turkey industry.

The turkey industry is moving towards the development of stud farms, but an essential condition is the existence of efficient methods to store semen. Much research has been done recently to determine the number of viable spermatozoa in an insemination dose needed for maintaining optimum fertility. Practical methods to determine the number of intact spermatozoa in semen, both before and after storage, are under development. It is now possible to store turkey semen for 6 to 24 h without appreciable loss in fertility and hatchability. Ideal experimental conditions for 48 h storage have not yet been fully determined. Development of new storage media, allowing the insemination of very low numbers of spermatozoa, may provide interesting possibilities regarding the use of elite sires.