The causes of reduced fertility with cryopreserved semen

Abstract

Cryopreserved mammalian semen is generally acknowledged to have an impaired fertility by comparison with fresh semen. The reduction arises from both a lower viability post-thaw and sublethal dysfunction in a proportion of the surviving subpopulation. The reasons for the loss of fertility are various. In this paper, factors affecting the proportion of survivors (e.g., cold shock susceptibility, cooling rate, diluent composition and osmotic stress) and factors influencing functional status of survivors (e.g., membrane stability, oxidative damage, membrane receptor integrity, nuclear structure) are briefly reviewed. The possible effects of cryopreservation on the role of spermatozoa in the early stages of embryogenesis are considered. In the light of this review, indications for new approaches for improving the performance of cryopreserved semen are offered.

Introduction

Cryopreservation of semen has long been seen as a means of benefiting the breeding of animals of agricultural importance, and has been recognised as contributing to the conservation of endangered species and to overcoming aspects of male infertility in humans. Nevertheless, with the possible exception of bull semen, a lower fertility is generally accepted as a consequence of cryopreservation. In this paper I want to offer a perspective on the causes of lower fertility.

Cryopreserved bull semen has been used commercially in dairy cattle for decades and conception results are now comparable or better than with natural mating. However, this is not so for the majority of mammalian species where fertility is clearly reduced by the cryopreservation protocol. Indeed, the losses from cryopreservation are compensated by the insemination of larger numbers of spermatozoa, and this is true for cattle semen as well as other species. The difference between species is explained by the extent of the compensation that can be achieved in cattle, where relatively few spermatozoa are required for good fertility, complete compensation can be achieved by inseminating of the order of 20 million total spermatozoa, still allowing for sufficient dilution of semen to make the process commercially viable. With other species many more spermatozoa are necessary to achieve reasonable fertility, and the losses associated with cryopreservation are such as to preclude the process being commercially viable. As a generalisation, some 40–50% of the population do not survive cryopreservation even with optimised protocols. When comparisons are made on the basis of similar numbers of motile (assumed viable) spermatozoa, results are still generally poorer than with fresh semen, indicating that even the viable subpopulation after cryopreservation is compromised. An approach to overcome the problem in some species has been to utilise surgical insemination permitting the inseminate to be introduced high in the reproductive tract to achieve a more satisfactory fertility, but at the higher cost of surgery.

This phenomenon can be explained by the need to have a sufficient number of fully competent spermatozoa capable of achieving fertilisation over the period when ovulation is likely to occur. The isthmus of the oviduct acts as a functional sperm reservoir providing a source of potentially fertilising spermatozoa over the ovulatory period Hunter, 1984, Hunter and Nichol, 1983. Only a minute proportion of spermatozoa introduced into the lower reaches of the female tract enter the oviducal reservoir, the majority being expelled through the vulva or phagocytosed in the tract. In the ampulla at the time of fertilisation, the sperm:oocyte ratio approaches unity (Hunter, 1996). The numerical and temporal competency of this sperm cohort depends on both the number and quality of spermatozoa introduced into the lower reaches of the tract. Apart from maternally-related aspects, conception at artificial insemination can be seen as a probability event determined by considerations of the inseminate. When the sperm number or quality in the inseminate is reduced fertility declines on an exponential curve (Fig. 1); normally, insemination is performed with sufficient competent spermatozoa to achieve results on the asymptote of this curve. Both the slope of the curve and the asymptote vary with individual sire and can be influenced by cryopreservation procedures (Amann and Hammerstedt, 1993). If the total number of fully functional spermatozoa in a cryopreserved inseminate falls below the number needed to achieve a high probability of fertilisation, then fertility is reduced.

When the inseminate is placed higher in the female tract than is normally the case with artificial insemination, fewer spermatozoa are required to achieve the same probability of fertility since a greater proportion will survive to colonise the oviduct. Thus, with sheep, intrauterine insemination is far more effective than posterior cervical insemination. Maxwell (1986) showed that only 20 million motile cryopreserved ram spermatozoa were required to achieve greater than 50% fertility, whereas it is recognised that cervical insemination requires 10 times that dose. If the insemination was made into the oviduct itself, less than 1 million spermatozoa were needed (Maxwell et al., 1993).

Another observation relating to cryopreserved semen is manifested with boar semen. In the pig, ovulation can occur over an extended period of oestrus such that spermatozoa may be required to survive up to 40 h in the oviduct. Waberski et al (1994) have noted that fertility with cryopreserved semen may be high providing the insemination is made in the period 4 h before ovulation. Outside this period, fertility with cryopreserved spermatozoa declined dramatically, but fresh semen maintained its fertility for a much longer period. Cryopreserved spermatozoa do not survive in the female tract compared with fresh spermatozoa.

In summary, the cryopreservation process results in reduced fertility compared with fresh semen. It has been shown that this arises from a combination of both loss of sperm viability and an impairment of function in the population of survivors. This situation needs to be borne in mind when strategies to improve the results are contemplated. We need to consider not only the cryopreservation protocol to optimise the number of survivors, but also the functional ability of the surviving population.