Fertility results after vaginal deposition of frozen-thawed buck semen diluted with two different extenders using one- or two-step procedures.

In two field trials (T1 and T2), the effect of two different extenders for buck semen was tested. Semen from six (T1) and seven (T2) bucks of the Norwegian Dairy Goat breed was diluted either in a milk-based extender containing egg yolk (M) or in a commercially available extender without egg yolk [Andromed(®) (A)]. Dilution in M was performed in a two-step procedure including centrifugation of the ejaculates and removal of the supernatant, while dilution in A was performed in one step. During the two trials (T1 and T2) 514 and 714, does, respectively, were artificially inseminated during natural oestrus, and the farmers performed the inseminations themselves after attending an artificial insemination (AI) training course. Vaginal insemination with 200 × 10(6) spermatozoa diluted in M resulted in a 25-day non-return rate (NRR) and kidding rate of 37.3% and 24.5%, respectively, while semen diluted in A resulted in 31.7% NRR and a kidding rate of 19.8% (T1). In T2, NRR and kidding rate for AI performed with semen diluted with M were 42.7% and 28.5%, respectively, while dilution in A gave 37.2% NRR and a kidding rate of 26.8%. There was no significant effect of extender in the two trials [T1:p=0.068 (NRR), p=0.148 (kidding rate), T2:p=0.096 (NRR), p=0.38 (kidding rate)], but farmer had a significant effect on the fertility parameters in both trials. In conclusion, the present studies may indicate that Andromed(®) is suitable for buck semen. However, more research is necessary to confirm the results and to improve the fertility of does after vaginal AI with frozen-thawed semen.

Vaginal deposition of frozen-thawed semen in Norwegian Dairy goats: comparison of single and double insemination with equal total number of spermatozoa.

In a field trial, a total of 472 Norwegian Dairy goats showing natural estrus were artificially inseminated with frozen-thawed semen. The farmers themselves performed vaginal deposition of 400 x 10(6) spermatozoa; one half of the does received two straws (200 x 10(6) spermatozoa/straw) at the same time (single AI), while the other half received two straws (200 x 10(6) spermatozoa/straw) 12 h apart (double AI). The commercially available extender Andromed was used for dilution. The does were housed at 15 different farms, and on average 31 does were inseminated per farm. Non return rates (NRR) and kidding rates after single insemination were 64.3% and 58.3%, respectively. Double inseminations resulted in a NRR of 62% and a kidding rate of 57%. No significant difference between single and double AI was seen in the study. This study indicates that single or double vaginal insemination with an equal total number of frozen-thawed spermatozoa (400 x 10(6)) can give acceptable fertility results in Norwegian Dairy goats. However, studies on reducing sperm numbers are called for to allow AI donor bucks to be used to their fullest potential.

Effect on field fertility of addition of gelatine, different dilution rates and storage times of cooled ram semen after vaginal insemination.

In a field trial, 633 ewes from 24 farms were inseminated vaginally using liquid semen (150 x 10(6) per dose) collected from 15 rams. The semen was either diluted with a milk-based extender (M), filled in 0.2 ml straws and stored for 12 or 24 h (M12, M24) or diluted with M but with the addition of gelatine, filled in 0.5 ml straws and stored for 12 or 24 h (G12, G24). The hypothesis was that a larger volume and the addition of gelatine would prolong the survival of the spermatozoa. The ewes, aged between 6 months and 5.5 years, were allocated into four groups and inseminated after natural oestrus by the farmers themselves with a dose of 150 x 10(6) spermatozoa. Inseminations in the groups (M12, M24, G12, G24) resulted in lambing rates of 69.6%, 63.6%, 69.4% and 58.3% (overall 65.2%), respectively. Farmer (p < .0002) had a significant effect on the lambing rate, while ram, age of the ewe and dilution rate/addition of gelatine/storage time had not. A pair-wise comparison of the lambing rates between the four groups showed that significant lower results were only achieved for G24 compared with M12. None of the other comparisons showed significant differences. In conclusion, a higher dilution rate of the AI-dose together with the addition of gelatine to the semen extender did not lead to improved fertility results after storage for 24 h when compared with standard AI-doses used in Norway.

Effect of different packages and freezing/thawing protocols on fertility of ram semen.

A field trial was performed in order to evaluate the effect on fertility of different straw types, freezing protocols (one- or two-step) and thawing procedures (35 degrees C and 70 degrees C) using frozen-thawed ram semen. A total of 791 Norwegian Crossbred ewes were artificially inseminated during natural oestrus with semen collected from nine mature and proven Norwegian Crossbred rams. A milk-based extender was used for dilution. The ewes were allocated into one of the following three groups based on the different straw types and thawing temperatures: medium straw (0.5 ml) thawed at 35 degrees C for 20 s (Med35), medium straw thawed at 70 degrees C for 8 s (Med70) and mini straw (0.25 ml) thawed at 35 degrees C for 15 s (Mini35). The semen to be frozen in mini straws was re-concentrated by centrifugation. Sperm number in each insemination dose was approximately 200 x 10(6) spermatozoa. The fertility results [as 25-day non-return rate (NRR)] for Med35, Med70 and Mini35 were 53.1%, 50.8% and 58.3%, respectively, and the lambing rates 49.8%, 46.8% and 53.8%, respectively. No significant main effects were seen for straw type/thawing temperature (p = 0.17), ram (p = 0.06) or age of the ewe (p = 0.18) on NRR or lambing rates (p = 0.19, p = 0.16 and p = 0.27, respectively). Both NRR and lambing rate differed significantly among farms (p < 0.0001).

Effect of vaginal and cervical deposition of semen on the fertility of sheep inseminated with frozen-thawed semen.

The effect of vaginal and cervical deposition of frozen-thawed semen on the fertility of sheep was tested in a field trial in which 543 Norwegian crossbred ewes aged between six months and five-and-a-half years from 10 farms were inseminated after natural oestrus. Cervical insemination with 200 x 10(6) spermatozoa resulted in 25-day non-return and lambing rates of 75.4 and 72.7 per cent, respectively, and vaginal insemination gave rates of 71.3 and 67.4 per cent; the cervical inseminations produced significantly higher lambing rates (P=0.04). There were significant differences between the lambing rates for different rams (P=0.006) and different farmers (P=0.003), and there was a significant interaction between farmer and deposition site (P=0.03). After vaginal insemination fertility was encouragingly high, but the results varied with the farmer, and different flock and management conditions.

Effect of cervical and vaginal insemination with liquid semen stored at room temperature on fertility of goats.

The effect of vaginal and cervical deposition of liquid semen stored at room temperature on the fertility of goats was tested in a field trial in which 217 Norwegian Dairy goats aged between 6 months and 7.5 years from 14 farms were inseminated after natural oestrous. Cervical insemination with 200 x 10(6) spermatozoa resulted in 25-day non-return and kidding rates of 87.0 and 78.0%, and vaginal insemination gave 85.5 and 74.3%, respectively. There was no significant difference between the cervical and vaginal inseminations (P = 0.59 for the 25-day non-return and P = 0.40 for the kidding rates). Farm had a significant effect on the 25-day non-return rate (P = 0.03) but not on the kidding rate (P = 0.07). There were no significant differences between the fertility rates for different bucks (P = 0.36 for the 25-day non-return and P = 0.15 for the kidding rates). Fertility results after vaginal insemination were encouragingly high. Vaginal insemination is a simple, less costly and time consuming technique compared to others, also bringing into focus the animal welfare aspects of the artificial insemination procedure. As the final goal is to establish a technique that could be applied similarly on a large scale by all farmers, vaginal insemination must be considered as a method that would simplify the use of liquid buck semen in Norway.

Effects of in vitro storage time and semen-extender on membrane quality of boar sperm assessed by flow cytometry.

The Norwegian AI company Norsvin has used the short-term semen-extender BTS to extend and store boar semen since the late 1980s. Fertility results have been consistent when extended semen has been used for AI within 3 days after collection, however, from a production and economic point of view it is preferable that semen stored for up to 5 days can be used. The aim of this study was to compare membrane quality of sperm stored in BTS for 3 days with sperm stored in the long-term semen-extenders Androstar, Mulberry III and X-cell for 5 days. Using a split-sample design, plasma membrane- and acrosome-integrity were assessed flow cytometrically by use of Yo-Pro-1 and PNA-FITC, and fluidity and phospholipid asymmetry of the membrane were assessed by use of MC540 and Annexin V-FITC. Due to observed sperm fragmentation in Androstar after Day 1, the data for Androstar were excluded from the analyses. After 5 days of storage, the membrane quality of X-cell-stored sperm was not statistically different from that of sperm stored in BTS for 3 days, while membrane quality of sperm stored in Mulberry III was statistically better on Day 5 compared to BTS on Day 3. In conclusion, Mulberry III and X-cell preserve sperm quality, as well as that of BTS on Day 3, for up to 5 days after collection.

Influence of boar and semen parameters on motility and acrosome integrity in liquid boar semen stored for five days.

Ninety ejaculates from a total of 76 AI boars were extended in Beltsville Thawing Solution (BTS). Boar identity, breed, weight of the ejaculate and sperm concentration were registered. Motility and acrosome integrity were assessed after storage at 16-18 degrees C for 6, 30, 54, 78, and 102 h. Storage time had a significant influence on both motility (p < 0.01) and acrosome integrity (p < 0.001). The Least Square Means for percentage of motility showed a small decline from 79.8% after 6 h of storage to 78.4% at 102 h. Motility at 78 and 102 h was significantly different from motility at 6 h (p < 0.05). The percentage of sperm cells with normal acrosomes declined throughout the experiment. The Least Square Means for 6, 30, 54, 78, and 102 h of storage were 93.9%, 90.6%, 88.0%, 84.8%, and 78.2%, respectively. The decrease in acrosome integrity from one storage time to the next was highly significant throughout the trial (p < 0.001). There was a significant influence of boar (p < 0.001) and sperm concentration (p < 0.01) on motility, while acrosome integrity was affected only by boar (p < 0.001). Breed of the boars and weight of the ejaculate did not influence the dependent variables.

Effect of deposition site and sperm number on the fertility of sheep inseminated with liquid semen.

The effect of the deposition site and the numbers of sperm on the fertility of sheep was tested in a field trial in which 1292 Norwegian crossbred ewes aged between six months and five-and-a-half years from 52 farms were inseminated with liquid semen after natural oestrus. Cervical insemination with 150 x 10(6) and 75 x 10(6) spermatozoa resulted in 25-day non-return rates of 63.7 and 56.1 per cent, and vaginal insemination gave non-return rates of 63.3 and 56.6 per cent, respectively. There was no significant difference between the cervical and vaginal inseminations, but the inseminations with 150 x 10(6) spermatozoa gave significantly higher non-return rates (P=0.004). There were significant differences between the non-return rates for different rams (P<0.0001) and farmers (P=0.0002) but the age of the ewe had no significant effect.

Seasonal changes in spermatogenic activity and in plasma levels of FSH, LH and testosterone, and the effect of immunization against inhibin in the male silver fox (Vulpes vulpes).

The cellular composition of the silver fox testis assessed by DNA flow cytometry and histological analysis exhibited marked circannual alterations. The proportion of haploid cells increased from late October to the breeding season in February, while that of diploid cells decreased and that of tetraploid cells fluctuated during the same period. Towards the end of March these changes were reversed. The seasonal variations in testicular histology paralleled the changes in distribution of cells from the different DNA populations. In August, 69% of the tubules contained spermatogonia as the only type of germ cell, while the remaining 31% also contained a few primary spermatocytes. In late October more than 50% of the tubules contained spermatocytes, and during the period of further activation from early December-February the seminiferous epithelium included round and/or elongated spermatids as well. In February, all tubules contained complete associations of germ cells, whereas in late March tubules with spermatogonia only and spermatogonia together with a few spermatocytes reappeared. In May, only such tubules could be found indicating total regression. Plasma concentrations of FSH and LH increased from early November, both gonadotrophins reaching maximum levels in December or early January, and then both declined during the second part of January, immediately prior to the actual breeding season. LH values showed a few smaller peaks in the beginning of June, whereas FSH levels were generally low until the next period of testicular reactivation. Testosterone concentrations were also low during most of the year but rose in November and December to reach a peak in January and a second peak in June. In animals immunized against inhibin the distribution of haploid, diploid and tetraploid cells did not deviate to any great extent from that in the controls, except in March when the immunized males had a markedly lower proportion of tetraploid cells, and in May, when they had a distinctly higher proportion of haploid cells. These findings were partly reflected by the histology. In the immunized animals, plasma FSH levels started to increase at approximately the same time but peaked higher and remained elevated almost 1 month longer than in the controls, whereas both the rise and decline in LH levels generally coincided with the variations in these animals, but the values were mostly higher. The testosterone profiles were similar to those in the controls except that the maximum values were also usually higher.

The spermatogenic cycle in the silver fox (Vulpes vulpes): frequency of the different stages prior to and during the breeding season.

In the silver fox the cycle of the seminiferous epithelium could be classified into eight characteristic stages defined on the basis of different, well-defined cell associations. The main criteria for the staging were the type of spermatogonia, the appearance of primary spermatocytes, the occurrence of meiotic figures and secondary spermatocytes and the shape and location of spermatids. In some cases more than one stage could be found within the same transverse tubular section. The average frequency of stages I to VIII was 25.2, 8.2, 9.0, 4.9, 16.2, 8.3, 10.7 and 17.5%, respectively. No significant difference was found between individuals sampled before and at the beginning and end of the breeding season. However, late in the season the migration of old spermatids and release of spermatozoa tended to be somewhat retarded, causing a slight increase in the duration and frequency of the last two stages of the cycle.

A preliminary study on the effect of dietary supplementation with cod liver oil on the polyunsaturated fatty acid composition of boar semen.

Eight mature Norwegian Landrace boars, of proven fertility and in routine semen production for AI, were fed individually with the same basic diet for 9 weeks. One group of 4 animals served as the control, the remaining 4 boars received a daily supplement of 75 ml cod liver oil (CLO-group). Fifteen consecutive semen samples were collected from each boar. The fatty acid composition of the semen was determined, and the content of the 15 most numerous fatty acids with a chain length longer than 12 carbon atoms was followed over time. In both groups, the proportion of 16:1n-7 decreased significantly, while 16:0 and 22:6n-3 (DHA) increased. By the end of the experiment, DHA had tended to increase and 22:5n-6 to decrease to a greater extent in the CLO-group. A significant difference between the groups was seen for one n-6 PUFA (22:4n-6), which remained unchanged in the control group but decreased in the CLO-group. No change was seen in docosapentaenoic acid (22:5n-3) and eicosapentaenoic acid (20:5n-3) was not found in any sample. These results indicate that CLO supplementation affects the fatty acid composition of boar semen. There were no significant differences in the non-return rates (4-25 days) between the two groups before, during or after the experiment.