J.K. Graham

EFFECTS OF CRYOPRESERVATION PROCEDURES ON SPERM MEMBRANES

Empirical approaches to semen cryopreservation have resulted in the production of young in a broad range of species. However, acceptable levels of fertility in most domestic animal species has not been achieved. In this review, an attempt has been made to describe the complexity of the sperm plasma membrane and the many steps in a cryopreservation procedure where membrane perturbations can occur. Improvement in sperm cryopreservation procedures will require a careful consideration of the complexity of the sperm plasma membrane, the interaction of its components and the influence of cooling, freezing and thawing on these interactions.

Assessment of sperm quality: a flow cytometric approach.

For many years, scientists have sought to develop laboratory assays that accurately predict the fertilizing capacity of a semen sample. This goal, however, has proven elusive and will most likely be very difficult to achieve, due to the complex nature of the problem. Part of the problem results from the many attributes that a spermatozoon must possess to fertilize an egg, and how laboratory assays can evaluate all of these attributes simultaneously. The percentage of motile sperm in a sample is most commonly used to evaluate semen quality. This assay, however, is not highly correlated with the fertilizing capacity of semen samples. One reason motion assays do not correlate well with fertility is that we are evaluating only one of many attributes that a sperm must possess to fertilize an oocyte. One of the problems of measuring multiple sperm attributes is the time and cost required. Using flow cytometric assays, multiple sperm attributes, including cell viability, acrosomal integrity, and mitochondrial function, can be measured simultaneously in sperm cells. In addition, the ability of sperm to undergo capacitation and the acrosome reaction, as well as the chromosomal integrity of sperm can be measured using flow cytometry. Flow cytometry permits us to evaluate 50,000 sperm in less then 1 min and at reasonable cost. Although flow cytometry is a powerful tool for evaluating many sperm attributes, it cannot evaluate all of the attributes a sperm cell requires to fertilize an oocyte. Therefore, laboratory assays are also being developed to evaluate the ability of sperm: (1) to bind to the oocyte, by evaluating the ability of sperm to bind to the perivitelline membrane of the hen egg in vitro; (2) to undergo an acrosome reaction in vitro, after treatment with membrane destabilizing compounds; and (3) to penetrate oocytes in vitro. When data from multiple sperm assays are used, higher correlations with the fertilizing potential of a semen sample is achieved. For example, in a study conducted utilizing five stallions, the percentage of motile sperm in semen samples correlated poorly with fertility (r(2)=0.22), however, when data for sperm motility, viability and penetration rates into zona-free hamster oocytes were utilized together, these data explained 72% of the differences in the fertility of the stallions (r=0.849; [Theriogenology 46 (1996) 559]). Armed with a battery of tests, which evaluate many different sperm attributes, researchers should be able to more accurately estimate the fertilizing potential of semen samples.

Cryopreservation of poultry sperm: the enigma of glycerol.

This review summarizes recent data for cryopreservation of poultry sperm and data establishing the contraceptive effect of glycerol. Successful cryopreservation protocols for bovine sperm are compared to the requirements for rooster sperm, with emphasis on glycerol-induced alterations in avian reproductive systems. It has been shown that molar concentrations of glycerol can affect (a) physical features of the cytoplasm (cytoplasmic organization and viscosity), (b) permeability and stability of the membrane bilayer(s), and (c) noncovalent attachment of proteins to the sperm surface. Perturbing effects of glycerol on sperm metabolism and the essentiality of maintaining bioenergetic balance during the temperature changes associated with any cryopreservation protocol are discussed. Emphasis is placed on the processes in avian reproduction that may be altered by interactions with glycerol. Finally, we discuss the potential value of using available genetic models (lines of roosters differing in the capacity of their sperm to survive a freeze-thaw cycle) to clarify and overcome damage to poultry sperm induced by cryopreservation.

Commercial semen freezing: Individual male variation in cryosurvival and the response of stallion sperm to customized freezing protocols ☆

One of the challenges for those attempting to cryopreserve stallion spermatozoa is dealing with the stallion to stallion variability in the cryosurvival of their semen. In the dairy industry, each bull stud, essentially utilizes a single cryopreservation technique, and bulls that produce sperm that do not cryopreserve well using that technique are replaced by other bulls. However, replacing stallions is unlikely to prove acceptable to the equine industry, where specific genotypes are desired. Instead, to increase the number of stallions that can be effectively utilized for cryopreserved semen production, it is likely that more than one method for cryopreserving sperm will be necessary. This manuscript reviews some of the processes involved in cryopreservation, how individual sperm physiology affects the ability to survive freezing and thawing, and how cryopreservation protocols can be customized to maximize sperm cryosurvival on an individual stallion basis.

Effect of Adding Cholesterol to Bull Sperm Membranes on Sperm Capacitation, the Acrosome Reaction, and Fertility

Abstract When cholesterol is added to sperm membranes before cryopreservation, higher percentages of motile and viable cells are recovered after thawing. However, because one of the first steps in sperm capacitation is cholesterol efflux from the sperm plasma membrane, adding cholesterol to enhance cryosurvival may retard sperm capacitation. These studies evaluated the ability of sperm treated with cholesterol-loaded cyclodextrins (CLC) to capacitate, acrosome react, and fertilize oocytes. Control (non-CLC-treated) and CLC-treated sperm were treated with heparin, dilauroylphosphatidylcholine (PC12), or calcium ionophore A23187 (A23187) to capacitate and induce the acrosome reaction. Sperm capacitation, assessed by an increase in intracellular calcium level, and acrosome-reacted sperm were measured using flow cytometry. Fresh CLC-treated sperm cells underwent capacitation and/or the acrosome reaction at rates different from control samples, and the differences detected were dependent on the method used to induce sperm capacitation and the acrosome reaction. After cryopreservation, however, CLC-treated and control sperm underwent capacitation and the acrosome reaction at similar rates regardless of the method used to induce capacitation and the acrosome reaction. The primary concern for CLC-treated sperm, however, is whether this treatment would affect in vitro or in vivo fertility. Adding either control or CLC-treated cryopreserved sperm to bovine oocytes in vitro resulted in similar oocyte cleavage rates and blastocyst formation rates. In addition, when inseminated into heifers, pregnancy rates for control and CLC-treated sperm were also similar. Therefore, treating bull sperm with CLC permits greater numbers of sperm to survive cryopreservation while preserving the fertilizing potential of each individual sperm.

Uterine horn insemination of heifers with very low numbers of nonfrozen and sexed spermatozoa

Abstract Experiments were conducted to determine 1) pregnancy rates of heifers inseminated with very low numbers of spermatozoa under ideal field conditions, and 2) pregnancy rates with low doses of sexed spermatozoa. In Experiment 1, semen from 3 Holstein bulls was extended to 1 × 105 or 2.5 × 105 sperm/0.1 ml; 2.5 × 106 total sperm/0.21 ml were used for the control. Semen was cooled to 5 °C, packaged into modified 0.25-ml French straws, and used 26 to 57 h after collection. Spermatozoa were inseminated 24 h after detection of estrus into the uterine horn of Holstein heifers ipsilateral to the ovary with the largest follicle, as determined by ultrasound 12 h after estrus was detected; side of ovulation was verified by detection of a corpus luteum (CL) by ultrasound 7 to 9 d post estrus. Pregnancy was determined 40 to 45 d post estrus. The side of ovulation was determined correctly in 262 of 286 heifers (92%), and pregnancy rates were nearly identical for ipsilateral and contralateral inseminations. Pregnancy rates were 48/118 (41%), 56/111 (50%), and 35/57 (61%) for 1 × 105, 2.5 × 105 and 2.5 × 106 sperm per insemination (P .05) among the heifers for the 3 AI technicians or the 3 bulls. In Experiment 2, freshly collected semen was transported from Lancaster, Pennsylvania to Beltsville, Maryland, and sorted into X- and Y-sperm populations based on DNA difference using a flow cytometer/cell sorter over a 6-h period. Sorting rates were about 100 sperm/sec of each sex at ~90% purity. Sorted spermatozoa were shipped ~2600 km by air, and in most cases cooled to 5 °C during shipping over 6 h in an Equitainer. Heifers were inseminated with 1 to 2 × 105 sorted X- or Y-spermatozoa in 0.1 ml within 9 to 29 h of sorting. The inseminate was either deposited into the uterine horn ipsilateral to the ovary with the largest follicle as determined by ultrasound at the time of insemination, or half of the inseminate was deposited into each uterine horn. None of 10 heifers became pregnant when inseminated with sexed spermatozoa shipped at ambient temperature. Of the 155 heifers inseminated with sexed spermatozoa cooled to 5 °C, 15 of 67 females (22.4%) inseminated 9 to 13 h post sorting calved, but only 2 of 78 (2.6%) inseminated at 17 to 29 h calved. Fourteen of the 17 calves bom (82%) were of the selected sex.

In vitro evaluation of sperm quality

This paper highlights selected laboratory analyses that are currently used to evaluate sperm, and describes why results from these assays do not consistently correlate with sperm fertility. Reasons for the disconnect between the two are due in part to the definition and reliability of the fertility data collected, to the complexity of the spermatozoon itself, to imprecision of some measurements, and to uncontrollable factors not associated to either the laboratory analysis or the sperm sample. Each sperm must possess a number of different attributes to fertilize an oocyte, and individual laboratory assays measure only one or a few of these attributes. Current and past data, correlating laboratory assay data with sperm fertility are presented in an effort to determine which types of assays are important to conduct and when to conduct them. Even though laboratory assay results do not allow accurate evaluation of the fertilizing potential of a semen sample, these assays are important to enable culling of poor quality samples.

Hysteroscopic insemination of low numbers of flow sorted fresh and frozen/thawed stallion spermatozoa.

The objective of this experiment was to determine the effects of flow cytometric sorting and freezing on stallion sperm fertility. A 2 x 2 factorial design was used to delineate effects of flow sorting and freezing spermatozoa. Oestrus was synchronised (July-August) in 41 mares by administering 10 ml altrenogest (2.2 mg/ml) per os for 10 consecutive days, followed by 250 microg cloprostenol i.m. on Day 11. Ovulation was induced by administering 3,000 iu hCG i.v. either 6 h (fresh spermatozoa) or 30 h (frozen/thawed spermatozoa) prior to insemination. Mares were assigned randomly to one of 4 sperm treatment groups. Semen was collected from 2 stallions with an artificial vagina and processed for each treatment. Treatment 1 (n = 10 mare cycles) consisted of fresh, nonsorted spermatozoa and Treatment 2 (n = 16 mare cycles) of fresh, flow sorted spermatozoa. Spermatozoa to be sorted were stained with Hoechst 33342 and sorted into X- and Y-chromosome-bearing populations based on DNA content using an SX MoFlo sperm sorter. Treatment 3 (n = 16 mare cycles) consisted of frozen/thawed nonsorted spermatozoa (frozen at 33.5 x 106 sperm/ml in 0.25 ml straws) and Treatment 4 (n = 15 mare cycles) of flow sorted frozen/thawed spermatozoa (frozen at 64.4 x 10(6) sperm/ml). Concentrations of sperm in both cryopreserved treatments were adjusted, based on predetermined average post-thaw motilities, so that each insemination contained approximately 5 x 10(6) motile spermatozoa. Hysteroscopic insemination of 5 x 10(6) motile spermatozoa in a volume of 230 microd was used for all treatments. Pregnancy was determined ultrasonographically 16 days postovulation. No differences were found (P>0.1) in the pregnancy rates for mares inseminated with fresh nonsorted (4/10 = 40.0%), fresh flow sorted (6/16 = 37.5%), frozen/thawed nonsorted (6/16 = 37.5%) and flow sorted frozen/thawed spermatozoa (2/15 = 133%). Pregnancy rates tended (P = 0.12) to be lower following insemination of frozen/thawed flow sorted spermatozoa. Further studies are needed with a larger number of mares to determine if fertility of flow sorted frozen/thawed spermatozoa can be improved.

Effect of seminal plasma on the motility of epididymal and ejaculated spermatozoa of the ram and bull during the cryopreservation process

Experiments were conducted to investigate the effect of seminal plasma on sperm motility during the cryopreservation process. Ejaculated and epididymal spermatozoa from the ram and the bull were washed by centrifugation and resuspended in either seminal plasma or a modified Tyrodes medium (TALP) prior to dilution in medium suitable for cryopreservation. Resuspension of washed ejaculated ram spermatozoa in seminal plasma resulted in higher percentages of motile spermatozoa than resuspension in TALP after the spermatozoa were cooled to 5 degrees C (52 vs 35%), and after thawing (14 vs 9%), respectively. Resuspension of epididymal ram spermatozoa in seminal plasma had no beneficial effect in maintaining sperm motility after cooling (78 vs 73%); however, seminal plasma was beneficial to epididymal ram spermatozoa after thawing (34 vs 3%), respectively. Resuspension of washed ejaculated bull spermatozoa in either seminal plasma or TALP had no effect on the percentage of motile spermatozoa after cooling to 5 degrees C (73 vs 75%) or after thawing (60 vs 60%), respectively. In addition, seminal plasma had no beneficial effect on the percentage of motile epididymal bull spermatozoa when compared with that of TALP-treated spermatozoa after cooling (75 vs 72%) or after thawing (66 vs 63%), respectively. Seminal plasma from different sires (ram and bull) affected epididymal sperm motility. The ability of sperm cells to withstand damage during cryopreservation, however, appears to reside in the sperm cells themselves, probably due to sperm cell composition.

In vitro capacitation of bovine spermatozoa: Role of intracellular calcium

The development of successful methods of in vitro fertilization for bovine oocytes has advanced the bovine as a model for reproductive technology. The discovery of heparin as a capacitating agent has made it possible for investigators to have an inexpensive, readily available supply of bovine gametes for experimentation in reproductive biotechnologies such as gene transfer and cloning. The central event that mammalian sperm must undergo before being able to fertilize an oocyte is capacitation. Although we have methods which lead to efficient in vitro fertilization, we still lack understanding about the molecular mechanisms of capacitation. While numerous events occur during capacitation, it appears that regulation of intracellular Ca2+ (Ca(i)) is one of the most important. We found that the influx of Ca2+ into sperm during the first 2 hours of incubation is critical to heparin-induced capacitation. This is a period during capacitation when Ca(i) has not yet increased. We propose that during capacitation, the initial influx of Ca2+ into sperm is used to fill an intracellular Ca2+ store located in the acrosome. We found that thapsigargin, an inhibitor of an acrosomal Ca2+-ATPase, can stimulate capacitated sperm to acrosome react, trigger the opening of a store-operated calcium channel in the plasma membrane and has greater effects on capacitated sperm compared to noncapacitated sperm. An increase in intracellular Ca2+ was also detected in the anterior sperm head during capacitation, suggesting the loading of the acrosome with Ca2+. These observations may be important in the development of new methods for capacitation and understanding the death of sperm after cryopreservation.