1. This study was designed to identify a suitable protocol for freezing turkey semen in straws exposed to nitrogen vapour by examining the effects of dimethylacetamide (DMA) or dimethylsulfoxide (DMSO) as cryoprotectant (CPA), CPA concentration, freezing rate and thawing rate on in vitro post-thaw semen quality. 2. Pooled semen samples were diluted 1:1 (v:v) with a freezing extender composed of Tselutin diluent containing DMA or DMSO to give final concentrations of 8% or 18% DMA and 4% or 10% DMSO. The semen was packaged in 0.25 ml plastic straws and frozen at different heights above the liquid nitrogen (LN2) surface (1, 5 and 10 cm) for 10 min. Semen samples were thawed at 4°C for 5 min or at 50°C for 10 s. After thawing, sperm motility, viability and osmotic tolerance were determined. 3. Cryosurvival of turkey sperm was affected by DMSO concentration. Freezing rate affected the motility of sperm cryopreserved using both CPAs, while thawing rates showed an effect on the motility of sperm cryopreserved using DMA and on the viability of sperm cryopreserved using DMSO. Significant interactions between freezing rate × thawing rate on sperm viability in the DMA protocol were found. 4. The most effective freezing protocol was the use of 18% DMA or 10% DMSO with freezing 10 cm above the LN2 surface and a thawing temperature of 50°C. An efficient protocol for turkey semen would improve prospects for sperm cryobanks and the commercial use of frozen turkey semen.

Cryopreserving turkey semen in straws and nitrogen vapour using DMSO or DMA: effects of cryoprotectant concentration, freezing rate and thawing rate on post-thaw semen quality.

Successful ultrarapid cryopreservation of wild Iberian ibex (Capra pyrenaica) spermatozoa.

A continuous decline in the number and range of capercaillie (Tetrao urogallus L.) in many European countries can be observed, mostly due to habitat destruction by human activity, unecological forestry management, and increased density of natural predators. Ex situ in vitro gene banks provide a unique opportunity to preserve the genetic material for future generations. Simple and effective cryopreservation methods for capercaillie semen are discussed. Semen was collected from seven males kept in the Capercaillie Breeding Centre at Forestry Wisla in Poland. Within five minutes after collection, ejaculates were diluted with EK diluent, then divided into two parts, and subjected to two freezing procedures: in pellets and in straws. In fresh semen, ejaculate clearness, viscosity, color and volume, as well as sperm concentration, motility and morphology, were evaluated, while in frozen-thawed semen only motility and morphology of sperm were determined. Fertilizing ability of thawed semen was examined for samples frozen in straws. Significant (P<0.05) differences between individual males were found in relation to the majority of fresh semen traits: ejaculate volume averaged 102.1 µL (varying from 49.0 to 205.0); average sperm concentration was 632.5 x106 mL-1 (178.8–1257.1); percentage of live normal cells varied from 39.2 to 70.3% (58.7% on an average); percentage of motile cells ranged from 76.0 to 85.7%) and motility parameters were male dependent, as well. Both cryopreservation methods had a negative effect on morphology and motility of frozen-thawed semen; however, the straw method yielded 60.7% and the pellet method 42.5% of live cells in total in thawed semen (P<0.05), while the number of live normal (intact) cells was similar (22.4 and 22.2%, respectively). Egg fertility varied between 77.8 and 91.7% (average 84.4%). Both freezing procedures seem to be effective in obtaining acceptable viability and high fertilizing potency of thawed sperm and can be used to create a gene bank of capercaillie semen.

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Simple and effective methods of freezing capercaillie (Tetrao urogallus L.) semen.

Successful preservation of capercaillie (Tetrao urogallus L.) semen in liquid and frozen states.

Abstract This review updates the current state of technologies available for turkey semen storage (hypothermic- liquid storage and cryopreservation), with special attention paid to cryopreservation. Liquid semen can be stored for up to 24 or 48 h at temperatures around 5°C, while cryopreservation allows long-term storage at -196°C. The possibility of using frozen turkey semen for artificial insemination (AI) would have practical benefits for turkey production. Reported fertility rates in response to AI using frozen/thawed semen range from 15.8 to 84.3%. Unsatisfactory fertility may be attributed to an inability of turkey spermatozoa to successfully survive the freezing/thawing process, and this, along with the high variability observed, makes this technique unacceptable for commercial breeding programs. There is therefore a need to standardize the whole freezing and thawing process to improve the post-thaw quality of turkey semen and minimize variability in results. Finding an efficient freezing protocol for turkey semen will allow for the creation of a sperm cryobank, improving current prospects for the commercial use of frozen turkey semen and also for the long-term conservation of the genetic diversity of this bird.

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Overview of Turkey Semen Storage: Focus on Cryopreservation – A Review

In Poland Capercaillie (Tetrao urogallus L.) is one of the most seriously endangered grouse species. The ability of semen collection and its utilization for Capercaillie female insemination would allow overcoming some fertility problems observed in captive-bred populations and thus reduce the rate of loss of genetic diversity. The present experiment was carried out on 13 individuals: eight males were kept with females and five alone. From each male, semen was collected four times, every second day, and overall semen appearance (color, viscosity), ejaculate volume, spermatozoa concentration, motility and morphology were examined. Ejaculates suitable for artificial insemination (AI) were obtained from 11 individuals. The volume of ejaculates varied from one drop (noted as 0.010 ml) to 0.180 ml, whereas spermatozoa concentration varied from 100 × 106 ml−1 to 1950 × 106 ml−1. The total amount of live spermatozoa for males kept with females varied from 82.0 to 98.3% (92.9% on average) and among them, from 38.7 to 82.0% were morphologically normal (67.6% on average), whereas for solitary males these values were the following: from 93.7 to 98.7 of total live (96.3% on average) and from 45.0 to 85.3% live normal cells (65.7% on average). No significant group effect was observed for above traits. Semen from males kept with females contained significantly (P<0.01) fewer cells with bulb head (12.2% vs. 21.6%), but higher numbers of bent neck spermatozoa (3.0 vs. 2.1%) and with other deformities (10.0 vs. 6.8%); however, for last two forms existing differences were not significant. Results obtained indicate the possibility of collecting valuable ejaculates from captive-bred Capercaillie, both kept with or without females, which makes possible the application of AI in order to increase the progeny number and gene exchange of this species across time and geographical distance. Zoo Biol 30:672–680, 2011. © 2010 Wiley Periodicals, Inc.

Characteristics of fresh semen of captive-bred capercaillie Tetrao urogallus L.

Artificial insemination (AI) is very helpful in solving the reproductive and biodiversity problems observed in small, closed avian populations. The successful production of fertilized eggs using AI is dependent on the collection of good quality semen. Two methods of male sexual stimulation and semen collection from captive kept capercaillie (Tetrao urogallus L.), one of the most seriously endangered grouse species in Europe, are compared in this study. Ejaculates were obtained either with the use of a dummy female or by the dorso-abdominal massage method. Differences in the individual responses of the males to the two methods of semen collection as well as in their semen quality were noted. Only sperm concentration (432.4 x 10(6) mL(-1) with dummy female and 614.5 x 10(6) mL(-1) for massage method) was significantly affected by capercaillie stimulation method. Sperm motility and morphology were not affected (P ≥ 0.05). Thus, for semen collection from captive kept capercaillie both methods can be used successfully. The dummy female can be an alternative to dorso-abdominal massage method, commonly used for semen collection from domesticated bird species.

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Comparative Examination of Capercaillie (Tetrao urogallus L.) Behaviour Responses and Semen Quality to Two Methods of Semen Collection.

Eggshell resorption, and embryonic mobilization and accumulation of calcium and metals in eggs of wild and captive Capercaillies Tetrao urogallus.

For many bird species in captivity best practice for incubation procedures have yet to be developed. This is hampered by a lack of cooperation between bird breeders or by a reluctance to experiment on valuable eggs. The last two problems may be solved by observation of natural incubation, which technology has made a lot easier. Many studies document incubation behavior: daily time spend in the nest, preferred hours of making absences, egg turning rate, incubation temperature. Such data is scientifically interesting but also allows for better protection of endangered species through development of captive breeding programmes. The Capercaillie (Tetrao urogallus) is a threatened species over much of its European range and various conservation actions are being taken to save populations from global or local extinction. Our study took place in Capercaillie Breeding Centre in Wisla Forestry District and describes nesting and incubation behaviour of Capercaillie females kept in captivity. Our aim was to better document the nesting behaviour by recording egg turning rate, and the time and length of incubation recesses. Time of day and day of incubation had no significant influence on recess length, but number of recesses was related with time of day with two peaks at 06:00 h and18:00 h. Egg turning activity was the greatest during the first and last two days of incubation but generally consistent throughout the rest of incubation. Captive hens spent less time outside of the nest than wild ones, their absences were shorter, rarer and took place mostly at the evening. However, differences in preferred absence hours were not as clearly marked as in case of wild birds. It is hoped that this information will help improve management practises to maximise the reproductive output of captive Capercaillie.

Zenon Rzońca

Papers

Successful preservation of capercaillie (Tetrao urogallus L.) semen in liquid and frozen states.

Characteristics of fresh semen of captive-bred capercaillie Tetrao urogallus L.

Comparative Examination of Capercaillie (Tetrao urogallus L.) Behaviour Responses and Semen Quality to Two Methods of Semen Collection.

Eggshell resorption, and embryonic mobilization and accumulation of calcium and metals in eggs of wild and captive Capercaillies Tetrao urogallus.

Nesting behaviour of Capercaillie (Tetrao urogallus) females kept in aviaries