The genetic improvement of agriculturally important species and disease control are of fundamental importance to the success of a sustainable agri-food industry. In this sense, artificial insemination (AI) is arguably the most important tool contributing to the advancement of modern animal production. Through AI, 1 ejaculate from a genetically superior male can be used to impregnate multiple females to maximize the distribution of agriculturally favorable genes. As well, AI eliminates physical contact between animals, thus limiting the spread of sexually transmitted diseases. Successful semen cryopreservation enhances these advantages of AI over natural breeding. Long-term storage facilitates semen transport over distances, permits the quarantine of semen, and enables extended use of superior germplasm, even after the sire’s death. For agriculturally important animals, semen cryopreservation is an established industry worldwide, particularly for dairy cattle. Genome resource banking to preserve the biodiversity of endangered species or valuable transgenic lines also would benefit from sperm cryopreservation. For many mammals; however, effective semen cryopreservation is not a reality because a large number of sperm are apparently infertile following freezing and thawing. Compared to fresh, 8 times more cryopreserved bovine sperm were required to achieve equivalent fertilization rates in vivo (Shannon and Vishwanath, 1995). Although AI in pigs with fresh, cooled semen is increasingly popular, the fertility of cryopreserved semen remains about half that of fresh semen (Crabo, 1991). Following either transuterine or oviductal insemination of superovulated ewes, conception rates and percentage of fer-

https://onlinelibrary.wiley.com/doi/pdf/10.1002/j.1939-4640.2000.tb03268.x

Semen cryopreservation in domestic animals: a damaging and capacitating phenomenon.

Cryopreservation of gametes and embryos of non-domestic species

Publisher Summary This chapter discusses the fundamental cryobiology of mammalian spermatozoa. The low motility of cryopreserved mammalian spermatozoa and the often lower conception rates may be due to the fact that procedures for cryopreservation of many mammalian cell types, including sperm, have evolved empirically. The primary assay of sperm function is the use of insemination and measurement of pregnancy initiation. The approaches to measuring sperm plasma membrane integrity include supervital staining and hyposmotic swelling. The other general approach to evaluating plasma membrane integrity is to assay the maintenance of membrane semipermeability by testing the cell's ability to change its volume when exposed to anisomotic conditions. Survival of cells subjected to cryopreservation depends not only on the presence of a permeating cryoprotective agent (CPA) but also on the concentration of the CPA. Viability of mammalian sperm is very sensitive to osmotic stress and the associated cell volume excursion. The optimization of CPA addition and removal procedures is also elaborated.

Fundamental Cryobiology of Mammalian Spermatozoa

I Comparative Overview of Mammalian Gametes- 1 The Coevolution of Mammalian Gametes- 2 Comparative Morphology of Mammalian Gametes- 3 A Comparison of Mammalian Sperm Membranes- 4 Mammalian Sperm Acrosomal Enzymes and the Acrosome Reaction- 5 Morphogenesis of the Mammalian Egg Cortex- 6 Comparative Structure and Function of Mammalian Zonae Pellucidae- II Studies on Mammalian Fertilization in Selected Species- 7 Fertilization of Marsupials- 8 Fertilization in Bats- 9 Fertilization in the Mouse: I The Egg- 10 Fertilization in the Mouse: II Spermatozoa- 11 Fertilization in the Golden Hamster- 12 Fertilization in the Rat- 13 Fertilization in the Guinea Pig- 14 Fertilization in the Rabbit- 15 Fertilization in Dogs- 16 Fertilization in Cats- 17 Fertilization in the Pig and Horse- 18 Bovine in Vitro Fertilization- 19 Fertilization in the Rhesus Monkey- 20 Fertilization in Man- III New Approaches for Studying Mammalian Gametes- 21 Micromanipulation of Mammalian Gametes- 22 The Use of Molecular Biology to Study Sperm Function- 23 Use of Molecular Biology to Study Development and Function of Mammalian Oocytes

A Comparative Overview of Mammalian Fertilization

Currently, more than 16,000 plant and animal species are imminently threatened by extinction, often as a direct consequence of anthropogenic influences. One of the measures to halt that process is genetic resource banking. This short review focuses on mammal sperm cryopreservation in combination with assisted reproduction techniques. It summarizes general problems, recent developments, and currently applied protocols and gives an overview of hitherto existing successes of assisted reproduction measures in wild animals in the light of conservation efforts.

Semen cryopreservation and the conservation of endangered species

Electroejaculates from 5 tigers were split and half of each was assayed fresh while the remainder was frozen and thawed before being assayed. Preincubation time, temperature and removal of seminal plasma were evaluated for their effect on in-vitro capacitation. Ability of spermatozoa to penetrate oocytes, as measured by the zona-free hamster egg-sperm penetration assay (SPA), was used as verification of capacitation. Results of the experiments with fresh semen indicate that: (1) preincubation time affects the fertilizability of tiger spermatozoa with 2 h appearing optimal, (2) a preincubation temperature of 37 degrees C results in significantly higher penetration rates than does a 22 degrees C treatment, and (3) tiger seminal plasma does not appear to contain decapacitation factors, as has been reported for several other species. Frozen semen experiments indicate that (1) frozen-thawed tiger spermatozoa must be removed from the environment of the semen extender before capacitation can take place, and (2) the freeze-thaw procedure results in a shortening of the required capacitation time.

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In-vitro induction of capacitation of fresh and frozen spermatozoa of the Siberian tiger (Panthera tigris).

Three experiments were designed to determine optimum conditions for capacitation of Siberian tiger (Panthera tigris altaica) sperm in vitro using the zona-free hamster egg sperm penetration assay (SPA) as a verification of capacitation. Sperm collected from a 9-year-old captive Siberian tiger were subjected to different in vitro washing conditions, preincubation times, and temperatures to induce capacitation. Sperm were able to penetrate zona-free hamster ova after 2 hours preincubation at 37°C but not at time 0. Preincubation at room temperature was not sufficient to prepare sperm for fertilization. The presence of seminal plasma during the 2-hour, 37°C preincubation did not affect the ability of tiger sperm to penetrate zona-free hamster eggs. The SPA can provide a means for evaluation of in vitro capacitation of Siberian tiger sperm.

G. A. Binczik

Papers

In-vitro induction of capacitation of fresh and frozen spermatozoa of the Siberian tiger (Panthera tigris).

In vitro capacitation of Siberian tiger spermatozoa