W.M.C. Maxwell

Storage of boar semen.

The problems, aspects and methods of liquid storage and freeze-thawing of boar semen are discussed and a review is given on examination of spermatozoa by the recent fluorescent staining methods.

Storage of ram semen.

Storage of ram semen in liquid and frozen state, the diluents used for both methods, processing, cooling, freezing and thawing of semen are reviewed. Factors influencing the fertility of stored semen and methods used for improvement are discussed, and fertility results of long-term frozen stored ram semen are also given.

Physiology of spermatozoa at high dilution rates: the influence of seminal plasma.

Extensive dilution of spermatozoa, as occurs during flow-cytometric sperm sorting, can reduce their motility and viability. These effects may be minimized by the use of appropriate dilution and collection media, containing balanced salts, energy sources, egg yolk and some protein. Dilution and flow-cytometric sorting of spermatozoa, which involves the removal of seminal plasma, also destabilizes sperm membranes leading to functional capacitation. This membrane destabilization renders the spermatozoa immediately capable of fertilization in vitro, or in vivo after deposition close to the site of fertilization, but shortens their lifespan, resulting in premature death if the cells are deposited in the female tract distant from the site of fertilization or are held in vitro at standard storage temperatures. This functional capacitation can be reversed in boar spermatozoa by inclusion of seminal plasma in the medium used to collect the cells from the cell sorter and, consequently, reduces their in vitro fertility. It has yet to be determined whether seminal plasma would have similar effects on flow cytometrically sorted spermatozoa of other species, and what its effects might be on the in vivo fertility of flow sorted boar.

Recent progress in the preservation of ram semen

Abstract The processing and storage of ram semen reduce the motility and disrupt the membrane integrity of spermatozoa. It is generally assumed that these changes are detrimental and are associated with a loss of fertilising capacity. Despite many years of empirical research that has established a variety of methods for the processing, storage and insemination of spermatozoa, fertility is generally lower after cervical insemination with stored than fresh semen. The components of the sperm cell responsible for this low fertility are not clearly understood. However, recent evidence suggests that while many spermatozoa remain motile after storage, the membranes of the motile cells are destabilised to the point where they may not survive further ageing in the female tract after cervical insemination. These membrane changes are similar to the capacitation and acrosome reaction of spermatozoa. Thus, stored spermatozoa may require less capacitation time in the female tract, and may readily fertilise oocytes if placed in their immediate vicinity, as with in vitro fertilisation, tubal or even intrauterine insemination. It may be possible to prevent or reverse some of these membrane changes, for example using antioxidants, and thus improve fertility following cervical insemination of sheep.

Frozen storage of ram semen I. Processing, freezing, thawing and fertility after cervical insemination

Abstract Research up to 1993 is reviewed. Various methods for processing, freezing and thawing of semen have been elaborated and their effect on survival and fertility of spermatozoa have been examined in vitro or in fertility tests. Diluents initially used for freezing bull semen had their limitations and were modified or replaced by new media. The freezing diluents investigated included citrate-sugar-, milk-, lactose-, saccharose-, raffinose-, trisbased and other media containing protective agents (mainly glycerol and egg yolk), and other substances including antioxidants. Satisfactory sperm survival rates were obtained with a number of diluents and different combinations of the factors studied. Lambing results after cervical insemination varied depending on the parameters examined in the freezing technology or at insemination, but were low in comparison with those obtainable with fresh diluted semen. As a consequence, cervical insemination with frozen-thawed semen had a relatively limited application in sheep.

Chlortetracycline analysis of boar spermatozoa after incubation, flow cytometric sorting, cooling, or cryopreservation

In this study, the effects of staining procedure with chlortetracycline (CTC) and method of analysis of boar spermatozoa after staining were examined. The hypothesis that incubation, flow cytometric sorting, cooling, and cryopreservation cause changes to boar sperm membranes which resemble capacitation and the acrosome reaction was also tested. Membrane status was evaluated by flow cytometry and by fluorescence microscopy after staining with CTC, and acrosome integrity was checked by flow cytometry after staining with FITC‐pisum sativum agglutenin and propidium iodide (PI). Flow cytometry was also used to assess viability (percentages of live and dead cells) of boar sperm after staining with SYBR‐14 and PI.

Frozen storage of ram semen II. Causes of low fertility after cervical insemination and methods of improvement

Abstract Research up to 1993 is reviewed. Freezing and thawing of ram semen causes ultrastructural, biochemical and functional damage to a significant proportion of spermatozoa. These changes are accompanied by a reduction in motility, impaired transport and decreased viability of spermatozoa in the female genital tract, and reduced fertility after cervical insemination. Methods used to improve the fertility after cervical insemination include increased concentration of spermatozoa in the inseminate, treatment of ewes with or addition to the semen of hormones, double and deep cervical inseminations. The most effective method is to increase the depth of deposition of frozen-thawed semen into the cervical canal. Recently developed transcervical insemination techniques achieve deep cervical insemination or even uterine deposition of semen. However, at present satisfactory and reliable lambing results are only obtainable by using intrauterine insemination by laparoscopy.

Birth of a male lamb derived from an in vitro matured oocyte fertilised by intracytoplasmic injection of a single presumptive male sperm

The developmental competence of in vitro matured ovine oocytes, cytoplasmically injected with single male or female chromosome-bearing sperm, was investigated. Eighty-five unsorted, 92 ‘female-sorted’ and 74 ‘male-sorted’ ram sperm were injected into in vitro matured sheep oocytes and, two to four hours later, placed into the oviducts of 28 oestrous sheep. The sperm were separated according to sex by analysis of their DNA content with a flow cytometer. One pregnancy was diagnosed by ultrasound after 55 days and a 3 kg male lamb was born after 150 days gestation. This lamb was derived from an oocyte injected with ‘male-sorted’ sperm.

In vitro and in vivo assessment of functional capacity of flow cytometrically sorted ram spermatozoa after freezing and thawing.

The effect of sex sorting and freeze-thawing on the viability and fertility of ram spermatozoa was investigated in the present study. Non-sorted (control) frozen-thawed spermatozoa had a higher motility and forwards progressive motility (FPM) than sorted frozen-thawed spermatozoa (60.9 +/- 2.9% v. 57.0 +/- 3.3% and 4.0 +/- 0.1 v. 3.5 +/- 0.1 FPM, respectively; P < 0.001) after incubation (6 h at 37 degrees C). Sorted and non-sorted (control) frozen-thawed spermatozoa had similar acrosome integrity (73.7 +/- 1.8% v. 75.2 +/- 2.1%, respectively) after thawing and incubation. A greater proportion of sorted spermatozoa displayed chlortetracycline staining patterns that were characteristic of capacitation (22.0 +/- 2.8%; P < 0.05) than non-sorted (control) spermatozoa (15.4 +/- 2.6% B pattern) before freezing. Overall, more sorted frozen-thawed spermatozoa showed patterns characteristic of being acrosome reacted (12.8 +/- 0.7%; P < 0.01) and less were uncapacitated (35.5 +/- 0.6%; P < 0.05) than non-sorted (control) frozen-thawed spermatozoa (7.7 +/- 0.8%; and 38.6 +/- 0.6% for AR and F pattern, respectively). Similar numbers of non-sorted (control) and sorted frozen-thawed spermatozoa migrated through artificial cervical mucus after 1 h (76.4 +/- 11.9 v. 73.9 +/- 11.9 spermatozoa, respectively). The distance travelled by the vanguard spermatozoon was also similar (56.9 +/- 7.8 v. 38.6 +/- 5.8 mm for control and sorted spermatozoa, respectively). Sorted and control frozen-thawed spermatozoa displayed a similar pattern of binding to, and release from, an oviduct epithelial cell monolayer (OECM), but sorted frozen-thawed spermatozoa were released more rapidly (P < 0.05) than non-sorted (control) frozen-thawed spermatozoa. The pregnancy rate was higher for ewes inseminated with 100 x 10(6) (commercial control) frozen-thawed spermatozoa (59%) than for 5, 10, 20 and 40 x 10(6) total sorted frozen-thawed spermatozoa (41% overall; P < 0.001). Insemination of 16 x 10(6) resulted in a higher pregnancy rate (31%) than 10(6) (17%; P < 0.05), but was similar to ewes that received 4 x 10(6) sorted frozen-thawed spermatozoa (24%). Time of insemination (54, 58 and 62 h after sponge removal) had no effect on pregnancy rate. Pregnancy in gonadotrophin-releasing hormone-treated ewes was affected by insemination dose (P < 0.05) but not sperm type (sorted and non-sorted) or ram. Pregnancy was higher after insemination of 40 x 10(6) than 5 or 20 x 10(6) non-sorted (control) or sorted frozen-thawed spermatozoa (70%, 33% and 35%, respectively; P < 0.05). Sorted frozen-thawed spermatozoa may have a shorter viability within the female tract than non-sorted frozen-thawed spermatozoa.

In vitro and in vivo developmental capacity of oocytes from prepubertal and adult sheep

Development to the blastocyst stage and survival following embryo transfer were assessed for oocytes obtained from prepubertal and adult sheep matured and fertilized in vitro. The rates of maturation, fertilization and cleavage in vitro did not differ significantly between oocytes from prepubertal and adult sheep. The proportion of cleaved zygotes reaching the blastocyst stage was significantly lower for oocytes derived from prepubertal than for those from adult sheep (15.4% and 34.1% respectively). There were no differences in the pregnancy rate and number of lambs born following transfer of blastocyst stage embryos derived from prepubertal and adult sheep to adult recipients. These data show that embryos derived from prepubertal lamb oocytes have reduced developmental potential in vitro but, of those which do reach the blastocyst stage, they have equal capacity to develop to term as embryos derived from adult sheep.