An effective method for freezing White Italian gander semen

Efficiency of freezing method, worked out for the White Italian gander semen was evaluated by comparing motility, morphology and fertilizing ability of spermatozoa in fresh and frozen-thawed semen. A part of pooled semen, collected from 25 White Italian ganders by dorso-abdominal massage was used immediately for artificial insemination of 10 geese (the control group) with a dose of 80 microl. This insemination was performed six times at weekly intervals. The remainder of the semen was diluted 1:0.5 (v/v) with EK diluent, equilibrated for 15 min at +4 degrees C, mixed with 6% (v/v) of dimethylformamide (DMF) and frozen to -140 degrees C at a rate of 60 degrees C/min. Frozen semen was thawed in a 60 degrees C water-bath and inseminated twice weekly in a dose of 100 microl (10 females of the experimental group, 12 inseminations were made). The freezing process affected spermatozoa motility and morphology, but had no effect on their fertilizing ability. Positive movement was observed in 50-60% of the spermatozoa in fresh semen and about 40% of the frozen-thawed cells. The average percentage of total live and live normal spermatozoa decreased due to freezing from 92.2 to 68.4% and from 34.7 to 14.1%, respectively. After the fresh semen insemination with average 12 million of the live normal spermatozoa per week average fertility was 88.24%; hatchability of set eggs was 80.88% and hatchability of fertile eggs was 91.67%. For frozen-thawed semen inseminated with average 9.5 million of the undamaged spermatozoa per week, the average fertility and hatchability rate was 83.78, 73.87, and 88.17%, respectively. Fecundity rates obtained after insemination with the frozen-thawed gander semen allow for the application of the freezing technique into breeding practice, in place of natural mating or to assist natural mating in periods of lowered fertility level.     

Evaluation of fresh and frozen-thawed semen of individual ganders by assessment of spermatozoa motility and morphology

Individual differences in gander Anser anser L. reaction to semen collection procedure, quality and quantity of fresh semen and its susceptibility to the freezing process are discussed. Semen was collected individually by dorso-abdominal massage, from 1-year old White Koluda ganders (n = 12) every 2-3 days. Ganders' reactions to massage were observed during the entire reproductive cycle (from 11 February to 13 June, from every male 40 semen collections were performed). For individual evaluation and freezing purpose semen was collected 13 times from every male. In the fresh semen, the following parameters were evaluated: ejaculate volume, color, density, blood or fecal contamination, motility, concentration and morphology of spermatozoa. Motility and spermatozoa morphology were evaluated in the frozen-thawed semen. Semen diluted in 2:1 ratio with EK diluent was frozen with 6% of dimethyl-formamide (DMF) to -140 degrees C at a rate 60 degrees C/min. Semen was thawed by placing the straws in a 60 degrees C water-bath for 4-5 s. Ten out of 12 ganders had from 67.5 to 100.0% positive reactions resulting in semen ejaculation. Significant (P < or = 0.01) differences in fresh semen quality of particular ganders were observed for all evaluated traits. In 1-year-old gander semen morphologically intact spermatozoa constitute only 27.8-45.2% of all cells. Therefore, the sperm quality factor (SQF), proposed by the authors, which includes ejaculate volume, sperm concentration and the percentage of live normal spermatozoa, seems to be a good predictor of gander semen fertilizing ability. The SQF of individual ganders varied from 7.7 to 11.5. The percentage of live normal spermatozoa in the frozen-thawed semen depended mainly on fresh semen quality. In relation to the fresh semen average from 57.2 to 63.2% of spermatozoa survived freezing process and from 23.9 to 38.5% remained morphologically intact.     

Effects of semen filtration and dilution rate on morphology and fertility of frozen gander spermatozoa.

Feces, urates or dirt originating from feathers often contaminate gander semen during collection, threatening its fertilizing ability. Seminal plasma used as a diluent has a similar effect, particularly on spermatozoa subjected to cryopreservation or short-term storage under refrigeration. The aim of the experiments was to evaluate the effects on spermatozoa motility, morphology and fertilizing ability after minimizing the influence of the contaminants by semen filtration or dilution prior to freezing. Pooled semen, collected twice a week from 9 White Italian ganders by dorso-abdominal massage, was divided into two parts. One sample was filtered and both were diluted in 1:1 or 1:0.5 (v/v) with EK diluent, equilibrated for 15 min at +4 degrees C, mixed with dimethyl-acetamide (DMA) in the final concentration 6% (v/v) and frozen to -140 degrees C in a computerized freezer, at a rate of 60 degrees C/min. In fresh and processed (filtered, freeze-thawed) semen were examined the spermatozoa motility and morphology, and fertilizing ability for freeze-thawed semen, both for unfiltered and filtered. In freeze-thawed semen no tangible differences due to experimental factors were observed in motility and percent of live spermatozoa in total. On average 35 to 42% of the spermatozoa survived the freezing process, but only 10 to 15% were normal, without any damage visible under the light microscope. The fertility of unfiltered freeze-thawed semen inseminated twice a week in a 0.2 mL dose (about 3 to 5 x 10(6) of live normal spermatozoa each) averaged 66.1% and hatchability of the set eggs 57.1 and 86.5% of the fertile eggs. The fertility obtained after the insemination with semen filtered prior to freezing was lower (64.3%), but hatchability was slightly higher (58.6 and 91.1% of set and fertile eggs, respectively). The duration of fertility for filtered semen was longer than that for unfiltered, 10 days after the last insemination the eggs were still fertile. The fertility results of freeze-thawed gander semen were very promising taking into consideration the small amount of inseminated live normal spermatozoa and it is possible to improve this result by increasing the number of spermatozoa in the insemination dose.     

The possibility of obtaining intergeneric hybrids via White Kołuda (Anser anser L.) goose insemination with fresh and frozen-thawed Canada goose (Branta canadensis L.) gander semen

The objective of the present experiments was to produce the intergeneric hybrids of domesticated and wild goose via artificial insemination with fresh and frozen-thawed semen. The experiments were carried out during two successive goose reproductive seasons, on eight five-year-old Canada Goose (Branta canadensis L.) males used as semen donors and 16 two-year-old White Ko?uda geese designated to fertility tests. Pooled semen was collected twice a week by the dorso-abdominal massage. In freshly collected semen, ejaculate volume, color, consistency, degree of fecal or blood contamination, spermatozoa concentration, motility, and morphology were evaluated. Part of the semen collected in the first year of the experiment (Experiment 1) was used for geese insemination with fresh semen, while the remainder was frozen. In Experiment 2 all samples were subjected exclusively to freezing procedure. Geese were inseminated once a week with fresh semen in a dose of 80 μl or 160 μl, and twice a week with frozen-thawed semen in a dose of 80 μl (160 μl per wk) or 100 μl (200 μl per wk). Eggs were set weekly and incubated up to hatching.The volume of ejaculates varied from 0.100 to 0.470 ml; spermatozoa concentration from 140 to 310 million ml⁻¹; progressive movement was observed in 40 to 60% of spermatozoa; the percentage of total live spermatozoa ranged from 69.3 to 92.0%, the highest percentage (34.0-68.3) was represented by live normal spermatozoa and those with bulb-head (13.3-41.0). Cryopreservation caused a decrease in percentage of motile cells to 30%; total live spermatozoa contribution by 27.2%p, including those live normal by 15.9%p (in relation to the fresh semen), bulb-head spermatozoa by 10.9%p, and increase (by 5.9%p) in number of spermatozoa with other deformations. Goose insemination 1×/week with fresh semen containing about 10.3 million live normal spermatozoa resulted in 66.7% of fertile eggs and with dose higher by 2.8 million spermatozoa (on average) the fertility increased by 20.9%p (up to 87.6% on average). Hatchability from set and fertile eggs was 55.9% and 83.9% vs. 66.3% and 75.6%, respectively. After twice a week insemination with frozen-thawed semen containing about 10.2 million live normal cells 58.2% eggs were fertile; hatchability from set eggs was 42.8% and from fertile eggs 71.7%, while insemination dose increase by 2.7 million spermatozoa per week caused a fertilization increase by 3.8%p (62.0% on average), this increase was not statistically significant, but hatchability from the fertile eggs (95.4%), was significantly (P < 0.05) higher.The use of AI with fresh semen in the creation of intergeneric hybrids of Canada goose males and White Ko?uda females allows a high level of egg fertility to be obtained. Furthermore, one limitation which is the short reproductive season of the Canada goose may be overcome by the use of cryopreserved semen.     

Cryogenic preservation of semen from the Aleutian Canada goose (Branta canadensis leucopareia)

Aleutian Canada geese (Branta canadensis leucopareia) were inseminated with frozen-thawed semen containing 6% or 7% dimethylsulfoxide (DMSO) resulting in 32 fertile eggs and 17 goslings; with 7% DMSO, 19 of 31 eggs were fertile. Beltsville Poultry Semen Extender (BPSE), adjusted to 270 ± 30 mOs and 7.5 ± 0.4 pH, was used to dilute semen samples and the DMSO before cryopreservation. About half of the live spermatozoa in the fresh semen (92.9 ± 2.5% live cells, laboratory studies; 87.3 ± 7.3%, insemination trials) survived the freeze-thaw process (46.7 ± 7.8%, laboratory; 33.3 ± 17.8%, insemination trials). Samples of frozen-thawed semen contained a greater percentage of bent spermatozoa (27.1 ± 8.4% of live cells) than fresh semen (14.4 ± 3.0% of live cells). Fecal- and urate-contaminated semen (a common problem when collecting goose semen) reduced the sperm motility score from 3.2 ± 0.6 to 2.7 ± 0.7 and number of live spermatozoa in frozen-thawed semen from 49 ± 9% to 24 ± 18%. Other variables examined that had less of an effect on semen quality included semen extenders, semen holding temperature, dilution and equilibration, relationship between hour of semen collection and level of semen contamination, and the relationship between season and sperm concentration.     

Comparison of three diluents for the storage of fresh bovine semen

In New Zealand, 95% of the semen used for artificial insemination in cattle is processed as liquid semen. Storage of liquid semen for up to 3 days in Caprogen) diluent enables a 10-fold reduction of the insemination dose, compared to frozen-thawed semen, without a reduction in fertility. In this Caprogen) diluent spermatozoa are stored under N2 gas in the presence of catalase. However, a new diluent (CEP-2), which was originally based on the biochemical composition of bovine cauda epididymal plasma, could become an appropriate alternative to Caprogen. In this study, the effect of addition of catalase to bovine spermatozoa stored for 6 days in CEP-2 diluent under aerobic and anaerobic conditions was evaluated and compared with a Tris diluent. Additionally, the quality and in vitro fertilizing capacity of fresh bovine semen stored for 6 days at 5 degrees C in the Triladyl, CEP-2 (without catalase and N2 gas) and Caprogen diluent were compared. Addition of 4.5 mg/mL catalase to CEP-2 diluent under aerobic and anaerobic conditions had no effect on sperm quality. Spermatozoa stored in CEP-2 diluent moved faster and straighter than spermatozoa stored in Triladyl or Caprogen diluent. The in vitro fertilization and polyspermy rates did not differ significantly between spermatozoa stored for 6 days at 5 degrees C in CEP-2 and Caprogen diluent, but were significantly lower for spermatozoa stored in Triladyl diluent. We can conclude that based on the in vitro results, the CEP-2 diluent is a better diluent than Triladyl and a good alternative to the Caprogen diluent for long term storage of fresh bovine semen at 5 degrees C. To confirm these promising in vitro results further in vivo experiments are required.     

Evaluation of a new diluent and different processing procedures for cryopreservation of ram semen

Ram semen was processed for freezing after initial dilution with a modified Tris-fructose diluent. Two aliquots were processed by cooling gradually to 5 degrees C, further dilution, equilibration and freezing in 0.5 ml straws either in pressurized liquid nitrogen (LN(2)) vapor (Method A) or on a block of dry ice (Method B). A third aliquot was cooled rapidly to 16 degrees C and then slowly to 5 degrees C, diluted further, equilibrated and frozen in straws in pressurized LN(2) vapor (Method C). The second dilution was carried out using a new diluent based on dextran-lactose. The diluted semen was equilibrated for 2 h before freezing. Semen was evaluated by artificial insemination (AI). The fertility of ewes bred by a double insemination with frozen-thawed semen processed by Methods A, B and C was 73% (n = 33), 67% (n = 30) and 80% (n = 30), respectively. In comparison, the fertility of ewes inseminated with fresh semen was 93% (n = 31). These preliminary data indicate an acceptable fertility can be achieved by AI with frozen-thawed semen processed using improved procedures.     

Detection of lipid peroxidation in frozen-thawed avian spermatozoa using C(11)-BODIPY(581/591)

The aim of this study was to perform flow cytometric analysis of C11-BODIPY581/591 oxidation in fowl and geese sperm as a marker for membrane lipid peroxidation (LPO) and to establish if the cryopreservation process would make sperm membranes more susceptible to oxidative stress. The experiment was carried out on 10 meat type line Flex roosters and 10 White Koluda® geese. The semen was collected two times a week, by dorso-abdominal massage method and pooled from 10 individuals of each species. Fowl semen samples were subjected to cryopreservation using the “pellet” method and Dimethylacetamide (DMA) as a cryoprotectant. Geese semen samples were cryopreserved in plastic straws in a programmable freezing unit with Dimethyloformamide (DMF) as the cryoprotectant. A fluorescent lipid probe C11-BODIPY581/591 provided with two double bonds that are oxidized during their contact with ROS, was used for the purpose of the assessment of the LPO in freshly diluted semen samples and frozen-thawed semen samples. This probe changes its color according to its state (non peroxidized: red; peroxidized: green). Flow cytometric analysis was used to monitor these changes. The White Koluda® geese fresh semen had a higher level of LPO than the Flex fresh semen (P > 0.01). The cryopreservation of fowl semen significantly (P > 0.01) increased the percentage of live and dead spermatozoa with lipid peroxidation. In frozen-thawed semen of White Koluda® geese the percentage of live spermatozoa with LPO significantly decreased (P > 0.05) whereas significantly (P > 0.01) higher level of dead cells with LPO was observed. There were significant differences between the two studied species. After thawing, the percentage of live and dead spermatozoa with lipid peroxidation was higher in fowl semen than in geese semen (P > 0.01). In conclusion, our data clearly indicate the existence of species specific differences in susceptibility of spermatozoa to the oxidation of PUFAs in the cell membranes, where such oxidation is caused by cryopreservation. This study shows that avian spermatozoa are vulnerable to radicals and frozenthawed sperm have higher level of LPO than fresh sperm. According to our observation, fowl semen is more susceptible to LPO than geese semen.     

A method of artificial insemination in captive White-tailed deer (Odocoileus virginianus )

Production of fawns by artificial insemination in captive White-tailed deer (Odocoileus virginianus ) has been accomplished by using frozen-thawed spermatozoa. The purpose of this study was to determine if frozen-thawed semen deposited at the posterior face of the os cervix could produce conception. Five hand-raised female White-tailed deer and one hand-raised male White-tailed deer were used over two breeding seasons 1984-1985 and 1985-1986. The vasectomized buck was ued to detect estrus in the does. The does were inseminated with frozen-thawed semen containing at least 100 million live normal cells with a 60% or higher motility. The artificial insemination catheters used in this study worked well, but due to the small size of the cervix, the catheter could only be passed up to the first cervical ring, the site at which the semen was deposited. Over two breeding seasons, nine does were inseminated with frozen-thawed spermatozoa; each doe was inseminated once each estrous cycle at one of the following times: 0, 6, 12, 18, 24 or 30 h. after detection of estrus. Of the nine does inseminated with frozen-thawed spermatozoa, six conceived and carried to term 11 healthy normal fawns, yielding an overall conception rate of 67%.     

Effect of freezing rate of ram spermatozoa on subsequent fertility in vivo and in vitro

Ram spermatozoa are most susceptible to damage during freezing between the temperatures of -10 degrees C and -25 degrees C. The objectives of the present study were to examine how freezing rate through this critical temperature zone affected the fertility of spermatozoa as assessed in vivo and in vitro. Semen from six adult rams was frozen at two different rates ("fast": 5 degrees C/min from +5 to -25 degrees C; "slow": 0.5 degrees C/min from +5 to -25 degrees C). In Experiment 1, semen from the fast and slow treatments was used to fertilize ovine oocytes that had been matured in vitro. Semen from the fast treatment yielded a higher cleavage rate (57% vs. 26%; P<0.001) and more blastocysts per oocyte (28% vs. 13%, P<0. 001) than slow-frozen. No correlation was found between fertilizing ability and viability as assessed by fluorescent probes. Experiment 2 was designed to establish the conception rates following both cervical and intrauterine insemination of frozen-thawed semen from the same bank of semen as used in Experiment 1. Ewes were superovulated with FSH and inseminated by laparoscopy with frozen semen. A significant difference was found in the number of fertilized ova following embryo recovery (81.4% vs. 39.3%; P<0.001). In a further study, 119 mature cull ewes were inseminated following a 12-day synchronization treatment with frozen semen by either intrauterine (laparoscopic) or cervical insemination. Insemination with fast-frozen semen resulted in a significantly higher pregnancy rate (P<0.05) irrespective of method of insemination. The data show that freezing rate affects the proportion of spermatozoa that retain their fertilizing ability post-thawing. However, once fertilization has occurred, development to the blastocyst stage is independent of freezing rate.     

The effect of DMA level on morphology and fertilising ability of Japanese quail (Coturnix japonica) spermatozoa

The effect of different levels (2, 4 or 6%) of DMA (dimethylacetamide) on the morphology and fertilising ability of unfrozen quail spermatozoa was evaluated. Semen was collected from 72 males kept individually in cages and randomly divided into four groups: Group I--control -- fresh undiluted semen (12 males) and three experimental groups (20 males each) - semen diluted 1:1 with Lake's extender and supplemented with 2% (Group II), 4% (Group III) or 6% (Group IV) of DMA (final concentration). Sperm morphology was evaluated at each step of semen preparation, i.e. in fresh and diluted semen, semen supplemented with DMA and semen that remained after insemination. For fertility tests, 36 females were divided into four groups (nine females each). Females in the control group were inseminated with 10 microl of fresh semen, in the experimental groups with 40 microl of diluted semen. Each stage of quail semen treatment had a deleterious effect on sperm morphology. The highest percentage of morphologically normal cells in semen evaluated after insemination, was observed in samples with 2% DMA, and the lowest--in samples with 6% DMA. Semen dilution and DMA addition significantly affected the fertilising potency of spermatozoa. Fertility of eggs collected from the control group (71.5% on average) was significantly higher (P相似文献   

Microscopic and flow cytometric semen assessment of Dutch AI-bucks: effect of semen processing procedures and their correlation to fertility

This study was done to determine the effects of processing techniques on the quality of semen from Dutch AI-bucks with the view on improving pregnancy rates after artificial insemination (AI) with liquid or frozen-thawed semen. Motility of spermatozoa was estimated under a microscope whereas the percentage live spermatozoa and the percentage live spermatozoa with intact acrosomes were determined by means of flow cytometry. Aspects of semen processing that were investigated are storage temperature of liquid semen (i), the effect of glycerol on liquid-stored semen (ii), removal of seminal plasma (iii) and type of extender (iv). The correlation between semen quality and fertility rates in inseminated does was also investigated. The percentage motile spermatozoa in semen stored in liquid form for 72 h progressively declined over time, irrespective of whether storage occurred at 4 or 18 degrees C. The percentage motile spermatozoa in semen stored at 18 degrees C was similar to that in semen stored at 4 degrees C if stored for 24 h but lower if stored for 48 h. Goats differ in the sensitivity of their spermatozoa to the deleterious effects of glycerol. Neither the removal of seminal plasma nor the type of extender had any effect on semen quality before freezing but semen frozen in a Tris-citric acid-glucose (TCG) buffer with egg yolk without removal of the seminal plasma had better quality after thawing than semen frozen in another diluent or after removal of seminal plasma. Remarkably no significant correlation between fertility and membrane integrity of spermatozoa could be found. Thus, although integrity assays for spermatozoa are useful to asses resistance to semen handling, the validity of these assays for predicting fertility is questioned.     

Effect of Dietary Selenium and Vitamin E on Ganders’ Response to Semen Collection and Ejaculate Characteristics

Compared to other domestic bird species, geese exhibit the lowest reproductive efficiency (poor semen quality, low egg production, and poor fertility and hatchability rates). From an economic perspective, it is a necessity of improve these reproductive traits. Studies have demonstrated that the essential trace element—selenium—plays key roles in testicular development and the maintenance of spermatogenesis. The aim of the present study was to determine the effect of feed supplementation with organic selenium and vitamin E on ganders’ response to manual semen collection and semen quality. Sixteen 3-year-old White Koluda ganders were randomly divided into two groups. The control group was provided commercial feed while the experimental group was provided with the same commercial feed supplemented with selenium (0.3 mg/kg) and vitamin E (100 mg/kg). The response of individual ganders from both groups to manual semen collection and the quality of the semen collected were evaluated. The supplements increased (P?≤?0.05) the frequency and decreased the time interval of a complete ejaculatory response of the ganders to manual semen collections (82.7 % supplement vs. 73.5 % control). Males from the supplemented group had significantly higher (P?≤?0.01; P?≤?0.05) ejaculate volumes, sperm concentrations, and percentages of viable sperm and lower percentages of immature sperm (spermatids). Lipids peroxidation, expressed in terms of the malondialdehyde concentration, was lower (P?≤?0.01) in semen of the supplemented group (0.172 nmol/50?×?10⁶) as compared to the controls (0.320 nmol/50?×?10⁶). Moreover, the duration of the reproductive period of the ganders in the experimental group was 1 week longer. The results show that supplemental dietary selenium and vitamin E improved both the ganders’ response to manual semen collection and semen quality. We conclude that such feed supplementation could lead to greater economic benefits through increased reproductive efficiency within the goose production industry.     

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

Experiments on semen collection and preservation were undertaken by Wroc?aw University of Environmental and Life Sciences and Forestry Wis?a, Poland to assist in the protection of the capercaillie (Tetrao urogallus L.) and to create an ex situ in vitro cryobank. Semen was collected from 11 captive-bred males, using dorsoabdominal massage. Ejaculates once obtained were diluted 3-fold at room temperature with EK diluent and then a number of them were stored at 4 °C for 18, 24, and 48 hours, while the remaining ejaculates were equilibrated with 6% dimethylacetamide and frozen by pipetting, drop-by-drop directly onto a liquid nitrogen surface. Frozen pellets were thawed at 60 °C in a water bath after 4 to 28 mo of storage. In total, 103 individually collected ejaculates (54 stored as liquid and 49 frozen in liquid nitrogen) were of appropriate value for further processing. The volume of ejaculates varied from 30 to 240 μL; spermatozoa concentration from 70 × 10⁶ mL⁻¹ to 1950 × 10⁶ mL⁻¹. The total amount of live spermatozoa in the fresh semen varied from 85.3% to 99.0%, of which from 41.1% to 85.3% were morphologically normal. Among morphologically abnormal forms, bulb-head (5.6% to 36.0%) and midpiece deformations (1.3% to 16.6%) were the most frequent. Dilution and semen storage up to 24 h at 4 °C did not affect the semen quality, as far as motility and sperm morphology are concerned. A significant (P < 0.05) decrease in total live (94.9 vs. 91.7%) and live normal cells (66.4 vs. 56.7%) was observed after 48 h. About 30% to 40% of spermatozoa remained motile. Cryopreservation significantly decreased (P < 0.05) the total number of live and live normal spermatozoa however, in relation to the fresh semen, their average content was 44.1% and 37.4%, respectively. Significant (P < 0.05) individual differences were observed in the quality of the fresh, liquid stored and the frozen-thawed semen assessed in terms of spermatozoa motility and morphology. After a single insemination with thawed semen containing 9.7 million live normal cells, 80% fertility and 100% hatchability were achieved. The obtained results indicate for the first time that there is the potential to use liquid stored and cryopreserved capercaillie semen to support conservation measures for the maintenance of genetic diversity, as well as to increase the number of reintroduced progeny of this endangered grouse species.     

Semen cryopreservation in Bactrian camel (Camelus bactrianus) using SHOTOR diluent: effects of cooling rates and glycerol concentrations

Experiments were conducted with a final goal of providing a suitable protocol for cryopreservation of Bactrian camel semen. In Experiment I, the effect of average cooling rate (slow cooling: 0.14 versus fast cooling: 0.55 degrees C/min) on the viability of chilled semen was evaluated. In Experiment II, the effect of different concentrations of glycerol (4, 6 and 8%) on the post-thaw viability of frozen sperm was investigated. In Experiment III, the efficiency of SHOTOR diluent was compared with IMV buffers for the cryopreservation of camel semen. Viability parameters including progressive forward motility (PFM), plasma membrane integrity and percentage of live spermatozoa were assessed. Progressive forward motility of sperm cooled at the faster rate was superior after incubating for 24h at 4 degrees C compared to that cooled at the slower rate (P<0.05). Post-thaw viability of Bactrian camel sperm was better using a final glycerol concentration of 6% compared to 4 and 8% (P<0.05). Progressive forward motility of frozen-thawed sperm was greater using SHOTOR diluent (29.9%) compared to IMV buffers (4.2%, P<0.05). In conclusion, semen cryopreservation in Bactrian camel is feasible when it is extended in SHOTOR diluent, cooled within 1h (average cooling rate: 0.55 degrees C/min) to 4 degrees C, and then exposed to glycerol, at the final concentration of 6%.     

Fertilizing capacity of equine spermatozoa stored for 24 hours at 5 or 20 degrees C

A breeding trial was conducted to evaluate the effect of in vitro storage time and temperature on fertilizing capacity of equine spermatozoa. Semen obtained from one stallion and diluted with skim milk-glucose extender was used to artificially inseminate 45 estrussynchronized mares. The mares were assigned to one of three treatment groups (15 mares per group): 1) insemination with fresh semen (collected within 0.5 h of use), 2) insemination with semen stored for 24 h at 20 degrees C or 3) insemination with semen stored for 24 h at 5 degrees C. The mares were inseminated daily during estrus, from the detection of a 35-mm follicle until ovulation, with 250 x 10(6) progressively motile spermatozoa (based on initial sperm motility of fresh semen). Semen samples (n = 35) were evaluated prior to insemination for percentages of total sperm motility (TSM), progressive sperm motility (PSM) and sperm velocity (SV). Single-cycle 15-d pregnancy rates. resulting from insemination with fresh semen, from fresh semen stored for 24 h at 20 degrees C or from semen stored for 24 h at 5 degrees C were the same (11 15 ; 73%). Mean diameters (mm) of 15-d embryonic vesicles were not different (P>0.05) among these three treatment groups (21.5 +/- 2.9, 19.6 +/- 2.6 and 20.5 +/- 3.6, respectively). Ten pregnant mares were aborted on Day 15 of gestation for use in another project. The pregnancy status of the 23 remaining pregnant mares was again determined at 35 to 40 d and 55 to 60 d of gestation. No pregnancy losses occurred during this time period. Mean TSM percentages were different (P<0.05) among the three groups: the fresh semen percentage was 89 +/- 2, semen stored for 24 h at 20 degrees C was 57 +/- 11 and semen stored for 24 h at 5 degrees C was 80 +/- 6. Similar differences were found for mean PSM and SV. Semen storage at either 20 or 5 degrees C for 24 h had no apparent effect on the fertilizing capacity of the extended semen samples; however, the reduction in all motility parameters tested was more dramatic in semen stored at 20 degrees C than that stored at 5 degrees C.     

Fertility of weaned sows after deep intrauterine insemination with a reduced number of frozen-thawed spermatozoa

The present study evaluates the effectiveness of the transcervical deep intrauterine insemination (DUI) with a reduced number of frozen-thawed boar spermatozoa in weaned sows. DUI was performed using a specially designed flexible device (length 180 cm, outer diameter 4mm, working channel 1.8mm, working channel's volume 1.5 ml) that was inserted through an artificial insemination spirette to cross the cervix lumen and moved into one uterine horn as far as possible. Spermatozoa diluted in 7.5 ml of BTS were flushed into the uterine horn by a syringe attached to the working channel. In Experiment 1, 111 hormonally treated (eCG/hCG) weaned sows were inseminated once using one of the following three regimens: (1) DUI with frozen-thawed spermatozoa (1000 x 10(6) cells per dose; n=49); (2) DUI with fresh semen (150 x 10(6) cells per dose; n=29, as control of DUI procedure); and (3) cervical insemination with frozen-thawed spermatozoa (6000 x 10(6) cells diluted in 100ml; n=33). No differences (P>0.05) were found for farrowing rates (77.55, 82.76, and 75.76, respectively) or litter sizes (9.31+/-0.41, 9.96+/-0.32, and 9.60+/-0.53 piglets born per litter, respectively) among the groups. In Experiment 2, DUI was performed on the spontaneous estrus in weaned sows (2-6 parity) with 1000 x 10(6) frozen-thawed (40 sows) or 150 x 10(6) fresh spermatozoa (38 sows). The farrowing rate of sows inseminated twice with frozen-thawed spermatozoa (70%) was significantly (P<0.05) lower than with fresh semen (84.21%). No significant difference (P>0.05) was found in litter size between frozen-thawed spermatozoa (9.25+/-0.23 piglets born per litter) and fresh semen (9.88+/-0.21 piglets born per litter). These preliminary results indicate that application of DUI provides acceptable fertility in weaned sows using a relatively low number of frozen-thawed spermatozoa.     

DNA fragmentation in chicken spermatozoa during cryopreservation

Semen cryopreservation is fundamental both for the practice of artificial insemination, and for the conservation of genetic resources in cryobanks; nevertheless, there is still not an efficient standard freezing procedure assuring a steady and suitable level of fertility in fowl, and consequently there is no systematic use of frozen semen in the poultry industry. This study examined changes in motility (CASA), cell membrane integrity (Ethidium Bromide (EtBr) exclusion procedure and stress test) and DNA fragmentation (neutral comet assay) in fowl spermatozoa before, during and after cryopreservation and storage at −196 °C. An optimized comet assay for chicken semen was studied and applied to the analyses. Semen collected from 18 Mericanel della Brianza (local Italian breed) male chicken breeders was frozen in pellets and thawed in a water bath at 60 °C. Measurements were performed on fresh semen soon after dilution, after equilibration with 6% dimethylacetamide at 4 °C (processed semen) and after thawing. Sperm DNA damage occurred during cryopreservation of chicken semen and the proportion of spermatozoa with damaged DNA significantly increased from 6.2% in fresh and 6.4% in processed semen to 19.8% in frozen-thawed semen. The proportion of DNA in the comet tail of damaged spermatozoa was also significantly affected by cryopreservation, with an increase found from fresh (26.3%) to frozen-thawed (30.9%) sperm, whereas processed semen (30.1%) didn't show significant differences. The proportion of total membrane damaged spermatozoa (EtBr exclusion procedure) did not increase by 4 °C equilibration time, and greatly and significantly increased by cryopreservation; the values recorded in fresh, processed and frozen semen were 2.9, 5.6, and 66.7% respectively. As regards the proportion of damaged cells in the stress test, all values differed significantly (7.1% fresh semen, 11.7% processed semen, 63.7% frozen semen). Total motility was not affected by equilibration (52.1% fresh semen, 51.9% processed semen), whereas it decreased significantly after cryopreservation (19.8%). These results suggest a low sensitivity of frozen-thawed chicken spermatozoa to DNA fragmentation, therefore it should not be considered as a major cause of sperm injuries during cryopreservation.     

First successful artificial insemination with frozen-thawed semen in rhinoceros

The first successful artificial insemination (AI) in a rhinoceros was reported in 2007 using fresh semen. Following that success, we decided to evaluate the possibility of using frozen-thawed semen for artificial insemination. Semen, collected from a 35-36 year old Southern white rhinoceros (Ceratotherium simum simum) in the UK was frozen using the directional freezing technique. This frozen semen was used in two intrauterine AI attempts on a 30 years old female rhinoceros in Hungary. The first attempt, conducted 30 days postpartum with an insemination dose of ∼135 × 10⁶ motile cells, failed. The second attempt, conducted two estrus cycles later with an insemination dose of ∼500 × 10⁶ motile cells, resulted in pregnancy and the birth of a healthy offspring. This represents the first successful AI using frozen-thawed semen in a rhinoceros, putting it among very few wildlife species in which AI with frozen-thawed semen resulted in a live birth. The incorporation of AI with frozen-thawed semen into the assisted reproduction toolbox opens the way to preserve and transport semen between distant individuals in captivity or between wild and captive populations, without the need to transport stressed or potentially disease carrying animals. In addition, cryopreserved spermatozoa, in combination with AI, are useful methods to extend the reproductive lifespan of individuals beyond their biological lifespan and an important tool for managing genetic diversity in these endangered mammals.     

Flow cytometric assessment of fresh and frozen-thawed Canada goose (Branta canadensis) semen

The present study was conducted to investigate spermatozoal membrane integrity, acrosome integrity, mitochondrial activity, and chromatin structure in fresh and frozen-thawed Canada goose (Branta canadensis) semen with the use of the flow cytometry. The experiment was carried out on ten, 2-year-old, Canada goose ganders. The semen was collected twice a week, by a dorso-abdominal massage method, then pooled and subjected to cryopreservation in straws, in a programmable freezing unit with the use of dimethyloformamide (DMF) as a cryoprotectant. Frozen samples were thawed in a water bath at 60 °C. The freezing procedure was performed ten times. For the cytometric analysis the fresh and the frozen-thawed semen was extended with EK extender to a final concentration of 50 million spermatozoa per mL. Sperm membrane integrity was assessed with SYBR-14 and propidium iodide (PI), acrosomal damage was evaluated with the use of PNA-Alexa Fluor^®488 conjugate, mitochondrial activity was estimated with Rhodamine 123 (R123), and spermatozoal DNA integrity was measured by the sperm chromatin structure assay (SCSA). The cryopreservation of Canada goose semen significantly decreased the percentage of live cells, from 76.3 to 50.4% (P < 0.01). Moreover, we observed the significant decrease in the percentage of live spermatozoa with intact acrosomes (P < 0.01), but we did not detect significant changes in the percentage of live spermatozoa with ruptured acrosomes. However, after thawing 50% of Canada goose live spermatozoa retained intact acrosomes. Furthermore, the percentage of live spermatozoa with active mitochondria was significantly lower in the frozen-thawed semen than in the fresh semen (P < 0.05). Nevertheless, after thawing the mitochondria remained active in almost 50% of live cells. In the present study, we observed no changes in the percentage of sperm with fragmented DNA after freezing-thawing of Canada goose semen. In conclusion, the present study indicates that even the fresh Branta canadensis semen might have poor quality, the cryopreservation of its semen did not provoke spermatozoal DNA defragmentation and half of the spermatozoa retained intact acrosomes and active mitochondria after freezing-thawing.