Conservative breeding ex situ in vivo is one of the most popular methods of creating genetic reserves. Unfortunately, keeping animals in small closed populations leads to inbreeding which reduces ...their reproductive capacity. The aim of the study was to characterize the sperm quality of 6 genetic groups of geese (northern and southern breeds) kept in Poland for many generations as genetic reserve flocks. Each breed was represented by 10 randomly selected 1-yr-old ganders, semen was collected 14 times, individually from each male, and the number of positive reactions (ended with ejaculation), semen volume, sperm concentration, and morphology were assessed. The obtained results showed a significant difference between breeds and individuals of the same group, both in males’ reaction and semen quantitative and qualitative traits. From the northern breeds 193 ejaculates were obtained in total (i.e., 45.9% of all attempts), from the southern breeds 242 ejaculates (57.6%). The volume of single ejaculate varied from 0.01 mL (one drop allowing only histological smear and sperm morphology evaluation) to 0.65 mL; sperm concentration varied from 23.0 × 106mL−1 to 2376.0 × 106mL−1; the amount of total live sperm was at a similar level in all breeds (89.6%–97.7%), while live normal cells ranged between 15.2% and 67.9% depending on breed and individuals. When keeping the genetic reserves ex situ in vivo, attention should be paid to the quality of semen and males that are poor in this respect should be eliminated, in order not to lead to an excessive weakening of the reproductive capacity of the flocks covered by the genetic resources protection program.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Artificial insemination is used in almost 95% of turkey reproductive flocks and is becoming more important in chickens, particularly broiler breeders, as well as in assisted reproduction of wild ...birds kept in breeding centers. Diluted semen is recommended for artificial insemination. Pooled semen samples collected twice a week by dorso-abdominal massage from 2 chicken lines: laying—ISA Brown (ISA-B) and meat type—Hubbard Flex (H-F) were divided into 5 parts: neat semen and diluted in 1:2 ratio with 4 extenders: basic EK; EK + 1 μg/mL organic selenium and 8 μg/mL vitamin E; EK + 10 mg/mL of royal jelly; and EK + 0.25 g/mL of lyophilized bovine colostrum. Diluted semen samples were evaluated after 15 min and then 24 h storage at 4°C. Sperm concentration, motility, motility parameters (with Sperm Class Analyzer), and morphology were evaluated in the neat semen, whereas in diluted and stored samples, the last 3 traits were determined. In case of both lines, dilution did not affect (P > 0.05) the number of live normal cells (78.0–81.1% in ISA Brown and 73.8–68.7% in Hubbard Flex) in relation to neat semen; however, bovine colostrum addition increased (P < 0.05) the percentage of bulb head sperm (5.7 vs. 10.0% and 12.1 vs. 17.6%, for ISA and Hubbard, respectively) and decreased sperm motility (67.4 vs. 92.9% and 67.3 vs. 98.5% for ISA and Hubbard). The 24 h storage of neat semen and semen diluted with colostrum caused (P < 0.05) the unfavorable changes in all evaluated traits and both chicken lines, whereas semen dilution with remaining extenders decreased the percentage of live normal cells (by 18.8–23.4% ISA and by 20.9–25.5% Hubbard) but did not affect sperm motility (81.5–87.6% for ISA and 81.1–96.6% for Hubbard). Sperm motility and motility parameters depended both on the extender and chicken line.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The aim of the present study was to determine the influence of chicken semen cryopreservation on sperm parameters, lipid peroxidation and antioxidant enzymes activities. Pooled semen from 10 Black ...Minorca roosters was used in the study. Semen samples were subjected to cryopreservation using the “pellet” method and dimethylacetamide (DMA) as a cryoprotectant. In the fresh and the frozen-thawed semen sperm membrane integrity (SYBR-14/propidium iodide (PI)), acrosomal damage (PNA-Alexa Fluor®488) and mitochondrial activity (Rhodamine 123) were assessed using flow cytometry. Malondialdehyde (MDA) concentration, catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities were determined in sperm cells and seminal plasma by spectrophotometry. All sperm characteristics evaluated using flow cytometry were affected by cryopreservation. After freezing-thawing, there was significant (P < 0.01) reduction in sperm membrane integrity, sperm acrosome integrity and mitochondrial activity. Following cryopreservation, MDA concentration significantly increased in chicken seminal plasma and spermatozoa (P < 0.01, P < 0.05). The CAT activity in seminal plasma significantly decreased (P < 0.05), while intracellular activity of this enzyme did not significantly change in frozen-thawed semen. In seminal plasma of frozen-thawed semen the significant increase (P < 0.01) in GPx activity was detected. Whereas GPx activity in spermatozoa remained statistically unchanged after thawing. The SOD activity significantly increased (P < 0.01) in cryopreserved seminal plasma with simultaneous decrease (P < 0.01) of its activity in cells. In conclusion, this is probably the first report describing the level of antioxidant enzymes in frozen-thawed avian semen. The present study showed that the activity of CAT, GPx and SOD in chicken semen was affected by cryopreservation, what increased the intensity of lipid peroxidation (LPO). Catalase appeared to play an important role in the sperm antioxidant defense strategy at cryopreservation since, opposite to SOD and GPx, its content was clearly reduced by the cryopreservation process. Change in the antioxidant defense status of the chicken spermatozoa and surrounding seminal plasma might affect the semen quality and sperm fertilizing ability.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Animal capture is essential in conservation and management programs, both for captive and wild species. This paper describes the effect of midazolam sedation in domestic and wild phasianids with ...respect to dorso-abdominal massage for obtaining semen samples, and on the quality of the sperm thus collected. Three experiments were performed. Experiment 1 - Determination, with respect to semen quality, of the optimum midazolam treatment for sedation in the domestic chicken. This involved sedating birds with either: 1) 2 mL of saline (control C), 2) 2 mg/kg of midazolam (low dose LD), 3) 6 mg/kg of midazolam (high dose HD), or 4) 6 mg/kg of midazolam and waiting 10 min before starting to massage (HD-10) (all performed in duplicate). The response time to the sedative (T1) was recorded, as were the times from the start of massage to ejaculation (T2) and its end (T3). The proportion of animals that showed stress during massage was noted via their waking, wing flapping, and the heterophil-lymphocyte ratio. The volume of all sperm samples obtained was determined, along with sperm concentration, motility, viability, and DNA integrity. Experiment 2 - The same determinations were made in captive red-legged partridges. Experiment 3 - The effect of the discovered optimum conditions (HD-10) for the above species was examined in the capercaillie (a wild species). Sperm quality was not affected by sedation with midazolam in any of the examined species, although the capercaillies and partridges were more nervous during semen collection than were the chickens.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The creation of genetic reserves of domesticated animal species and breeds almost become a necessity in the recent years, but there is a question what is the value of semen of males kept ex situ in ...vivo as gene conservation flocks. Presented studies assessed the response to semen collection by dorso‐abdominal massage and the quantitative and qualitative semen characteristics of six goose breeds (Pomorska, Garbonosa, Kuban, Landes, Roman and Slovakia) covered by the genetic resources’ protection programme. Fourteen semen collection attempts were performed per male. In each breed there were ganders with low and high sensitivity to massage. The most positive reactions were stated in Pomorska ganders (67.9%) and the least in Kuban breed (52.60%). Individual male evaluation showed that only in three breeds (Pomorska, Garbonosa and Kuban) there were individuals showing 100% susceptibility to semen collection, in some breeds only one to four positive reactions (ending with ejaculation) were noted. Results obtained indicated breed and male effect on analysed semen traits, with the exception of sperm motility. The highest number of live normal sperm (44.2% on average), sperm concentration (530 × 106 ml−1), the highest Semen Quality Factor (92.9) and sperm motility (50.30%) were found in semen of Kuban ganders, while the lowest values of these traits (28.7%; 230 × 106 ml−1; 11.4 respectively) in Slovakia ganders. The lowest sperm motility (38.3%) was observed in ganders of Roman breed, but comparing to the other breeds existing differences were not significant. Significant differences in sperm morphology between individual ganders were also observed.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
The Western capercaillie (Tetrao urogallus) is a specific bird species, which, despite its very broad distribution and large global population size, is highly endangered in many Western and Central ...European countries. According to the species situation, in many countries (including Poland), breeding and reintroduction programmes have been started. One of the most complex and large-scale reintroduction programmes was started in Bory Dolnośląskie Forest, and the Capercaillie Breeding Centre in Wisła Forest District was used as one of the sources of individuals for reintroduction. As genetic tools provide essential knowledge about species biodiversity, which is crucially important during the breeding process and reintroduction, both captive and reintroduced grouse populations were genetically analysed. We were particularly interested in genetic diversity of the individuals in both populations and the genetic relationship between them, as well as between them and other capercaillie representatives from their current range. To fulfil these goals we determined nine microsatellite loci along with a fragment of the mitochondrial control region. Genetic diversity parameters were moderate to high compared to populations from other Central and Western European countries. Both populations were clustered into three distinct genetic clades based on microsatellites. Phylogenetic analysis placed all mitochondrial haplotypes we revealed in the Eurasian clade. The present results will play an important role as they will help to preserve and maximize genetic diversity in captive populations, and will provide a basis for future monitoring of the reintroduction process.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Males of Muscovy duck (Cairina moschata) are mainly used for mule duck production via artificial insemination of females originated from wild mallard duck (Anas platyrhynchos); therefore, the ...quantity and quality of drake semen play a crucial role. The assessment results of male reaction to sexual stimulation by dummy female and basic semen characteristic (ejaculate volume, sperm concentration and morphology) of 12 individually kept Muscovy drakes carried out during the entire reproductive season are described. The male and period of the reproductive season effect on scored semen traits are documented. In total, 792 individual semen collections and evaluations were performed. The average of positive reaction in the entire reproductive season varied from 90.6% in December and April to 50.0% in July, while for individual males, it varied between 97.1% and 29.0%. Throughout the season, the ejaculate volumes ranged from 0.05 to 2.45 ml, sperm concentration from 0.15 × 109 ml−1 to 4.44 × 109 ml−1, total number of live spermatozoa from 68.0% to 100% and live normal (properly formed, with any deformations) from 51.0% to 99.0%. Our study indicates the necessity of male breeders pre‐selection before the onset of the reproductive season, and the need to leave an appropriate number of males to ensure adequate amount of semen for female insemination, especially when using Muscovy drakes (Cairina moschata) for interspecies crossing with Anas platyrhynchos ducks.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
The aim of this study was to assess the spermatozoal viability, acrosome integrity, mitochondrial activity, and DNA status in the frozen-thawed fowl semen with the use of flow cytometry. The ...experiment was carried out on 10 sexually adult roosters of meat type line Flex. The semen was collected three times a week by dorso-abdominal massage method, then pooled and subjected to cryopreservation using “pellet” method and Dimethylacetamide (DMA) as a cryoprotectant. For cytometric analysis the fresh and frozen-thawed semen was extended with EK diluent to a final concentration of 50 million spermatozoa per mL. Sperm membrane integrity was assessed with dual fluorescent probes SYBR-14 and propidium iodide (PI). Acrosomal damages were evaluated using phycoerythrin-conjugated lectin PNA from Arachis hypogaea. The percentage of live spermatozoa with functional mitochondria was estimated using Rhodamine 123 (R123) and PI. The spermatozoal DNA integrity was measured by sperm chromatin structure assay (SCSA). The freezing-thawing process decreased the viability, mitochondrial activity in the chicken sperm and increased the percentage of dead cells with ruptured and intact acrosomes, and also the percentage of spermatozoa with fragmented DNA. In conclusion, the present study indicates that fluorescent staining and flow cytometry may be useful for assessment of the changes of fowl semen quality caused by cryopreservation process. This technique allows precise examination of spermatozoa functional characteristic in a very short time.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Avian semen dilution with appropriate extender allows to prolong the fertilizing ability of sperm stored in vitro. In the present study, the impact of extenders and time of storage on morphology of ...Muscovy duck (Cairina moschata) drake semen were examined. Semen was collected twice a week, using male stimulation by a female method, from 12 adults (29 weeks old) drakes kept individually in cages, under controlled environmental conditions. Freshly collected, pooled ejaculates were divided into three part: neat undiluted sample, and diluted 1:1 with Schramm (SCH) or Watanabe (W) extender and stored at 4°C. Morphological examination of all samples was conducted after dilution and then, after 3 and 6 hr of storage. The storage of undiluted semen caused decrease (p ≤ .01) in live morphologically normal sperm, from 79.73% in the freshly collected ejaculates to 55.75% and to 12.12% after 3 and 6 hr of storage, respectively (average calculated for the entire reproductive season). In the semen diluted with Schramm's extender the adequate values attained 86.84, 79.65 and 61.66%, and using Watanabe extender 84.77, 83.58 and 75.25%, respectively. The period of semen storage and the type of extender caused significant (p ≤ 0,05; p ≤ 0,01) changes in sperm morphology. The longer period of storage contributed to the decrease in number of morphologically normal sperm, whereas their content in Watanabe extender after 3 and 6 hr of storage was higher (p ≤ .01) than in semen diluted in Schramm extender.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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