Artificial insemination in domestic cats (Felis catus)

Artificial insemination (AI) in cats represents an important technique for increasing the contribution of genetically valuable individuals in specific populations, whether they be highly pedigreed purebred cats, medically important laboratory cats or endangered non-domestic cats. Semen is collected using electrical stimulation, with an artificial vagina or from intact or excised cauda epididymis. Sperm samples can be used for AI immediately after collection, after temporary storage above 0 degrees C or after cryopreservation. There have been three and five reports on intravaginal and intrauterine insemination, respectively, and one report on tubal insemination with fresh semen. In studies using fresh semen, it was reported that conception rates of 50% or higher were obtained by intravaginal insemination with 10-50x10(6) spermatozoa, while, in another report, the conception rate was 78% after AI with 80x10(6) spermatozoa. After intrauterine insemination, conception rates following deposition of 6.2x10(6) and 8x10(6) spermatozoa were reported to be 50 and 80%, respectively. With tubal insemination, the conception rate was 43% when 4x10(6) spermatozoa were used, showing that the number of spermatozoa required to obtain a satisfactory conception rate was similar to that of cats inseminated directly into the uterus. When frozen semen was used for intravaginal insemination the conception rate was rather low, but intrauterine insemination with 50x10(6) frozen/thawed spermatozoa resulted in a conception rate of 57%. Furthermore, in one report, conception was obtained by intrauterine insemination of frozen epididymal spermatozoa. Overall, there have been few reports on artificial insemination in cats. The results obtained to date show considerable variation, both within and among laboratories depending upon the type and number of spermatozoa used and the site of sperm deposition. Undoubtedly, future studies will identify the major factors required to consistently obtain reliable conception rates, so that AI can become a practical technique for enhancing the production of desirable genotypes, both for laboratory and conservation purposes.     

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

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 × 10(6) ml(-1) to 1950 × 10(6) 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.     

Storage of poultry semen

Methods of semen collection and artificial insemination (AI) in poultry, requirement for diluents, methods of liquid and frozen storage of avian semen and evaluation of spermatozoa after storage for fertilizing ability are reviewed. Frozen storage of semen from non-domestic birds is also briefly discussed.     

Effect of insemination dose on pregnancy rate in mares

Different insemination doses have been used for artificial insemination(AI) in horses. Since the insemination dose can affect the pregnancy rate, it is important to ensure that an adequate dose be used regardless of the type of inseminationprotocol used. The aim of this study was to find out if it is possible to decrease the insemination dose from 500 x 10(6) progressively motile spermatozoa to 300 x 10(6) progressively motile spermatozoa and still maintain an acceptable pregnancy rate when using extended fresh semen. Thirteen stallions of known fertility and a well-defined group of 64 mares were used in the study. The mares were randomly assigned to 1 of 2 insemination groups. Examination for pregnancy was performed by ultrasonography per rectum approximately 16 d after the last insemination. When using an insemination dose of 300 x 10(6) progressively motile spermatozoa the pregnancy rate per cycle was 75%. With an insemination dose of 500 x 10(6) progressively motile spermatozoa the pregnancy rate per cycle was 64%. There was no significant difference in the pregnancy rate between the 2 insemination doses (P = 0.341). We conclude that when using fresh extended semen it is unlikely that an insemination dose of 300 x 10(6) progressively motile spermatozoa would yield a lower pregnancy rate than a dose of 500 x 10(6) progressively motile spermatozoa if stallions with good quality semen are selected.     

Artificial insemination of sheep: the effect of semen diluents containing egg yolk on the fertility of ram semen.

Results from an artificial insemination (AI) experiment revealing the effect of semen dilutents containing egg yolk on the fertility of ram semen are presented. Ram semen was diluted 30-fold in buffered glucose-saline solution containing . 375, 1.5, or 6% v/v egg yolk and a portion of each was used soon for the AI of ewes or was incubated at 35 degrees C for 1 hour prior to AI. Some of the semen collection was used undiluted for AI of 10(8) spermatozoa/dose. All diluted samples were reconcentrated by centrifugation so that each dose was 10(8) spermatozoa in a volume of 100 mcl. 1146 ewes were inseminated. Fertility was assessed from 28 to 45 day nonreturns to estrus and nonreturn rates (NRRs) were expressed as percentages for the various treatments. Undiluted semen (controls) revealed 69%, semen used soon after dilution, .375% yolk in dilutent 58%, 1.5% yolk 50%, 6% yolk 42%; diluted semen incubated for 1 hour before use, .375% yolk 49%, 1.5% yolk 51%, and 6% yolk 39%. NRR was significantly depressed by dilution (p less than .001) and by increasing amounts of egg yolk (p less than .01) in the dilutent. Incubation of diluted semen before AI caused a small fall in NRR.     

Quality and fertility of cooled-shipped stallion semen at the time of insemination

Stallion semen processing is far from standardized and differs substantially between AI centers. Suboptimal pregnancy rates in equine AI may primarily result from breeding with low quality semen not adequately processed for shipment. It was the aim of the study to evaluate quality and fertility of cooled-shipped equine semen provided for breeding of client mares by commercial semen collection centers in Europe. Cooled shipped semen (n = 201 doses) from 67 stallions and 36 different EU-approved semen collection centers was evaluated. At arrival, semen temperature was 9.8 ± 0.2 °C, mean sperm concentration of AI doses was 68 ± 3 x 10⁶/ml), mean total sperm count was 1.0 ± 0.1 x 10⁹, total motility averaged 83 ± 1% and morphological defects 45 ± 2%. A total of 86 mares were inseminated, overall per season-pregnancy rate in these mares was 67%. Sperm concentration significantly influenced semen motility and morphology at arrival of the shipped semen. Significant effects of month of the year on volume, sperm concentration and total sperm count of the insemination dose were found. The collection center significantly influenced all semen parameters evaluated. Semen doses used to inseminate mares that became pregnant had significantly higher total and progressive motility of spermatozoa and a significantly lower percentage of morphological semen defects than insemination doses used for mares failing to get pregnant. Results demonstrate that insemination with semen of better quality provides a higher chance to achieve pregnancy. Besides the use of stallions with good semen quality, appropriate semen processing is an important factor for satisfying results in artificial insemination with cooled-shipped horse semen.     

Boar spermatozoa encapsulated in barium alginate membranes: a microdensitometric evaluation of some enzymatic activities during storage at 18 degrees C

The customary dilution of boar semen for subsequent artificial insemination (AI) procedures damages the cell membrane of spermatozoa, resulting in a loss of enzymes and other cytoplasmic contents and acrosomal reactions. We encapsulated non-diluted boar semen in barium alginate membranes to optimize AI procedures and to improve the functional integrity of spermatozoal membranes during storage. The percentage of non-reacted acrosomes (NRA) and measurements of enzyme leakage (cytochrome c oxidase (COX), lactate dehydrogenase (LDH), and glucose-6-phosphate dehydrogenase (G6PDH)) were used as indices of the functional status of diluted, unencapsulated and encapsulated spermatozoa, stored for 72 h at 18 degrees C. Enzymatic activity was assessed in situ by microdensitometry, and non-reacted acrosomes were microscopically determined by staining. The percentage of acrosome integrity and the intracellular enzymatic activities during storage were different for unencapsulated and encapsulated semen. Semen dilution caused a rapid decline in enzymatic activities and concomitant acrosomal reactions. Encapsulated spermatozoa had significantly higher acrosome integrity (77% versus 55%; P < 0.01 after 72 h) and an overall higher in situ enzymatic activity. For cytochrome c oxidase and lactate dehydrogenase the greatest differences between encapsulated and unencapsulated spermatozoa were present after 72 h whereas for glucose-6-phosphate dehydrogenase significant differences were found within 24h of storage. The encapsulation process maintains a better preservation environment for boar spermatozoa and could be a promising, innovative technique to improve storage of these cells.     

The dependence of the growth rate and meat content of young boars on semen parameters and conception rate

Boars have a decisive impact on the progress in pig production, however, there is no recent information about the optimal growth parameters during the rearing period for modern breed later used in artificial insemination (AI) stations. Therefore, the objective of the research was to conduct semen parameter and conception rate analyses on the basis of growth rate and meat content assessments made during the rearing of AI boars of different genotypes. The study was carried out between 2010 and 2014 and included 184 boars in five breed combinations: 46 Polish Large White, 50 Polish Landrace, 27 Pietrain, 36 Duroc×Pietrain and 25 Hampshire×Pietrain. Boars were qualified by daily gains and meat content assessment (between 170 and 210 days of life). A total number of 38 272 ejaculates were examined (semen volume (ml), spermatozoa concentration (×10⁶ ml⁻¹), total number of spermatozoa (×10⁹) and number of insemination doses from one ejaculate (n)). The fertility was determined by the conception rate (%). Semen volume, spermatozoa concentration and conception rate (P<0.01), followed by the total number of spermatozoa and insemination doses (P<0.05) were characterized by the highest variability in relation to breed of boars. The effect of daily gains was reported for spermatozoa concentration, number of insemination doses, conception rate (all P<0.01) and total number of spermatozoa (P<0.05). The peak of growth for spermatozoa concentration, total number of spermatozoa, insemination doses and conception rate was achieved for 800 to 850 g gains. Meat content affected semen volume, number of insemination doses and conception rate (P<0.05). Rearing boars while maintaining daily gains at the 800 to 850 g level and 62.5% to 65% meat content helps AI stations to increase the efficiency and economic profitability, and the number of insemination doses to increase by up to 300 doses/boar within a year. The analyses of growth parameters may help increase the efficiency and economic viability of AI stations.     

Laparoscopic insemination technique with low numbers of spermatozoa in superovulated prepuberal gilts for biotechnological application

New biotechnologies, such as sperm-mediated gene transfer (SMGT), spermatozoa freezing and spermatozoa sorting have improved the possibilities to produce animals with desirable features. The main problem associated with these technologies is the scarce availability of spermatozoa for insemination. The objective of this study was to develop a laparoscopic insemination (LI) technique in gilt that allows the use of low semen doses resulting in high fertilization rates (FR) and minimal distress to the animal; the efficiency of this technique was compared to conventional artificial insemination (AI). Ten gilts were inseminated 36 h post hCG treatment near both utero-tubal junctions (UTJ) with 1.5 x 10(9)spermatozoa/5 mL per horn and 10 gilts (C) underwent conventional AI. Embryos were collected either at two to four cell stage (LI, n = 5; C, n = 5) for determination of fertilization rate or at day 6 for evaluation of developmental competence (LI, n = 5; C, n = 5). LI gilts showed a slightly higher FR than control animals. In a second trial, 24 gilts underwent LI with varying doses (1.5 x 10(8), 1.5 x 10(7), 1 x 10(7), 5 x 10(6) or 1 x 10(6)) of semen. Two to four stage embryos were collected and FR was evaluated in each tube. FR obtained with the lowest dose was significantly different from that with other dosages (P < 0.05). Embryos were cultured in vitro to blastocyst stages (percentage of blastocysts: 79.2 +/- 3.6%). In a third trial, five gilts were inseminated with semen processed by SMGT technique; both FR (86.1 +/- 9.9%) and transgene protein expression were satisfactory. In conclusion, this study shows that LI can be a useful tool for reducing doses of insemination, without affecting the efficiency of fertilization; this technique could have a wide range of biotechnological applications.     

Reproductive response of ewes synchronized with different lengths of MGA treatments in intrauterine insemination program

A total of 415 fat tailed ewes were randomly assigned to two groups to assess the effect of duration of melengestrol acetate (MGA) (9 versus 12d) administration on reproductive parameters associated with laparoscopic artificial insemination. At the end of MGA treatment, ewes in each group were subdivided and inseminated with one of two different insemination doses (10×10(7) or 20×10(7) sperm per 0.5 ml insemination dose) of fresh diluted semen. Inseminations were carried out 11-18 h after first detected estrus. Ewes were screened for their return to oestrus from 10 to 21 days post AI and inseminated at their returned oestrus. Pregnancy diagnosis was done from approximately 55 days after insemination in both synchronized and return estrus. For short (9-day) and long (12-day) term MGA treated groups, estrus rates were 62% versus 89% (P<0.0001), respectively. Ewes (n=115) that returned to estrus were inseminated (7-11h after estrus detection) with fresh diluted semen at different doses (20×10(7) or 40×10(7) or 60×10(7) sperm per 0.5 ml insemination dose). Pregnancy rates were 41% and 44% for short term and long term MGA treated ewes, respectively. Pregnancy rate of ewes which returned to oestrus was 53.4%. There was a significant (P<0.05) increase in pregnancy rates (38-52% for 11-16 h; 63% for 17-18 h) when insemination was held at 17-18 h after first detected estrus following MGA treatments. Pregnancy rates were found to be similar in ewes inseminated with 10×10(7) (36%) or 20×10(7) (47%) motile spermatozoa at first AI, and 20×10(7) (44%) or 40×10(7) (59%) or 60×10(7)(48%) at second AI. It was concluded that short term MGA treated ewes were recorded with lower estrus rates but was similar to pregnancy rates with long term MGA treatment. Acceptable pregnancy rates were achieved in MGA induced estrus when insemination is conducted at 17-18 h after estrus onset and with 20×10(7) sperm per insemination dose.     

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 × 10⁶ spermatozoa; one half of the does received two straws (200 × 10⁶ spermatozoa/straw) at the same time (single AI), while the other half received two straws (200 × 10⁶ 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 × 10⁶) 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.     

Impaired semen quality of AI bulls fed with moldy hay: a case report

The daily quality control of semen at a Finnish artificial insemination (AI) bull station is based on subjective motility and sperm morphology of young bulls entering the semen collection program. Semen quality dropped suddenly in autumn 1998. During 5 consecutive months, the number of rejected ejaculates and discarded frozen semen batches due to poor motility increased, and the number of all forms of abnormal spermatozoa increased. However, for the accepted ejaculates, a 60 day nonretum rate was normal. The summer of 1998 in Finland was rainy, and the hay used in the AI station was visibly moldy. Immunoassay and gas chromatography-mass spectrometry (GC-MS) detected Fusarium mycotoxins HT-2 and T-2, but no zearalenone in the hay. Occurrence of mycotoxins such as T-2 and HT-2 in the moldy hay coincided with, and may have been responsible for the impaired semen quality in AI bulls. This case report will draw the attention to the possible hazards when feeding moldy hay.     

Development of an artificial insemination protocol in llamas using cooled semen

The objective of this study was to design an AI protocol using cooled semen to obtain pregnancies in the llama. Each raw ejaculate was subdivided into four aliquots which were extended 1:1 with: (1) 11% lactose-egg yolk (L-EY), (2) Tris-citrate-fructose-egg yolk (T-F-EY), (3) PBS-llama serum (S-PBS) and (4) skim milk-glucose (K). Each sample reached 5°C in 2.5 h and remained at that temperature during 24 h. Percentages of the semen variables (motility, live spermatozoa) in ejaculates and samples cooled with L-EY were significantly greater than those obtained when cooling with the other extenders; therefore this extender was used (1:1) for all inseminations. Females were randomly divided into four groups (A-D) according to insemination protocol. Group A: females were inseminated with a fixed dose of 12 × 10(6) live spermatozoa kept at 37°C. Group B: females were inseminated with a fixed dose of 12 × 10(6) live spermatozoa, cooled to 5°C and kept for 24 h. Group C: females were inseminated with the whole ejaculate (variable doses), cooled to 5°C and kept for 24 h. These groups (A-C) were inseminated between 22 and 24 h after induction of ovulation. Group D: females were inseminated with the whole ejaculate (variable doses), cooled to 5°C, kept for 24 h and AI was carried out within 2 h after ovulation. Pregnancy rates were 75%, 0%, 0% and 23% for groups A, B, C and D respectively. These results indicate that it is possible to obtain llama pregnancies with AI using cooled semen and that the success of the technique would depend on the proximity to ovulation.     

Uterine secretion from mares with post-breeding endometritis alters sperm motion characteristics in vitro

Uterine secretion was collected from five normal mares during estrus by the use of a tampon. In subsequent estrus cycles, mares were inseminated with 1 x 10(9) spermatozoa from a stallion of known fertility, and uterine secretion was collected randomly at 6, 12, and 24 hours after insemination. All mares had negative endometrial cytology before insemination. At the time of uterine secretion sampling, semen was collected from two stallions and extended with Kenney's extender to a concentration of 50 x 10(6) spermatozoa/mL. Extended semen was diluted 2:1 with uterine secretion; semen extender; and centrifuged uterine secretion (noncellular). Samples were kept at room temperature and sperm motion characteristics (corrected motility (CMOT), progressively motile spermatozoa (PMS), and mean path velocity (MPV) were evaluated using a computer-assisted semen analyzer every 40 minutes for a total of 4 hours. Sperm motion characteristics of spermatozoa were significantly better when incubated in semen extender compared to uterine secretion (P < 0.05). The CMOT and PMS were significantly better in uterine secretion collected before, compared to after AI with the lowest values observed in samples collected at 12 hours after breeding (P < 0.05). Sperm motion characteristics of spermatozoa incubated in centrifuged uterine secretion was only slightly suppressed compared to spermatozoa incubated in semen extender, suggesting that the altered motion characteristics were mostly due to the presence of polymorphonuclear neutrophils (PMNs) in the samples. It was concluded from this study that spermatozoa can survive in inflamed uterine secretion, but that sperm motion characteristics in vitro are altered.     

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.     

Some effects of the composition of inseminated semen and the site of its deposition on fertility in Gallus domesticus

It is most probable that during natural copulation the semen of the fowl is ejaculated into a shallow position in the vagina of the hen, but during the commercial application of artificial insemination it is generally considered necessary to evert the distal vagina and deposit semen to a depth of at least 5 cm to produce optimal fertilisation of the succession of eggs laid daily by a female for a week post-insemination. Aspects of the artificial insemination technique in relation to the types of semen that are obtained from the male fowl artificially are re-appraised in relation to their effect on fertility. It was confirmed that a smaller number of spermatozoa (50 × 10⁶) than is normally used in commercial practice (>80 × 10⁶) produced good fertility, even when inseminated within 0.5 cm of the vaginal opening in the cloaca. The results were achieved whether or not glucose was present in the inseminate. When semen was deposited in the cloaca, a better fertilisation rate was obtained if ductus-deferens semen was diluted with transparent fluid, which is produced by tumescent tissue in the cloaca during semen collection. However, the same advantageous effect was shown by dilution with synthetic aqueous fluids with and without glucose. The likely role of transparent fluid during natural copulation is discussed. On the basis of the number of spermatozoa found to maintain good fertility by artificial insemination, only 10 μl semen would be required to be ejaculated into each hen during copulation. This may account for the well-known ability of the male fowl to copulate frequently in a day, because the small volume of semen would be replenished, naturally, very quickly in the ductus deferens.     

Effects of different artificial insemination techniques and sperm doses on fertility of normal mares and mares with abnormal reproductive history

The effects of different artificial insemination (AI) techniques and sperm doses on pregnancy rates of normal Hanoverian breed mares and mares with a history of barrenness or pregnancy failure using fresh or frozen-thawed sperm were investigated. The material included 187 normal mares (148 foaling and 39 young maiden mares) and 85 problem mares with abnormal reproductive history. Mares were randomly allotted into groups with respect to AI technique (routine AI into the uterine body, transrectally controlled deep intracornual AI ipsilateral to the preovulatory follicle, or hysteroscopic AI onto the uterotubal junction ipsilateral to the preovulatory follicle), storage method of semen (fresh, frozen-thawed), AI volume (0.5, 2, 12 ml), and sperm dose (50 x 10(6) or 300 x 10(6) progressively motile sperm (pms) for fresh semen and 100 or 800 x 10(6) frozen-thawed sperm with >35% post-thaw motility). The mares were inseminated once per cycle, 24 h after hCG administration when fresh semen was used, or 30 h for frozen-thawed semen. Differences in pregnancy rates between treatment groups were analyzed by Chi-squared test, and for most relevant factors (insemination technique, mare, semen, and stallion) expectation values and confidence intervals were calculated using multivariate logistic models. Neither insemination technique, volume, sperm dose, nor mare or stallion had significant effects (P > 0.05) on fertility. Type of semen, breeding mares during foal heat, and an interaction between insemination technique, semen parameters, and mares did have significant effects (P < 0.05). In problem mares, frozen semen AI yielded significantly lower pregnancy rates than fresh semen AI (16/43, 37.2% versus 25/42, 59.5%), but this was not the case in normal mares. In normal mares, hysteroscopic AI with fresh semen gave significantly (P < 0.05) better pregnancy rates than uterine body AI (27/38, 71% versus 18/38, 47.3%), whereas in problem mares this resulted in significantly lower pregnancy rates than uterine body AI (5/15, 33.3% versus 16/19, 84.2%). Our results demonstrate that for problem mares, conventional insemination into the uterine body appears to be superior to hysteroscopic insemination and in normal mares, the highest pregnancy rates can be expected by hysteroscopic insemination.     

Low-dose insemination--why, when and how

The typical dose for insemination into the uterine body of the mare is > 300 x 10(6) progressively motile spermatozoa (PMS) and an insemination dose of > 200 x 10(6) PMS is recommended for frozen-thawed semen. Low-dose insemination techniques allow for a drastic reduction in the numbers of spermatozoa required to achieve pregnancy. Acceptable pregnancy rates can be achieved with doses ranging from 1 to 25 x 10(6) PMS in volumes ranging from 20 to 1000 microL. Two techniques have been described: hysteroscopic insemination and transrectally guided deep horn insemination using a pipette. Similar pregnancy rates can be attained by either method when 5 x 10(6) PMS are used. Hysteroscopic insemination may provide an advantage when the dose is 1-3 x 10(6) PMS. These techniques have the potential to make more efficient use of frozen-thawed or sex-sorted semen from certain stallions. The use of low-dose insemination to improve fertility of infertile stallions warrants further investigation.     

Birth of the first mithun ( Bos frontalis) calf through artificial insemination

The study describes the standardization of a suitable semen cryopreservation protocol for the first time in mithun (Bos frontalis) and birth of the first mithun calf through artificial insemination. The semen samples were collected from adult bulls through the rectal massage method and cryopreserved in liquid nitrogen using tris-egg yolk-glycerol diluent. The diluted semen samples were packaged in 0.50 ml straws and kept at 5°C for 4 h for equilibration. Following the equilibration, the straws were frozen into liquid nitrogen vapour for 10 min and then plunged into liquid nitrogen for storage. It was observed that the progressive motility (%) decreased significantly (P < 0.01) in cryopreserved semen (43.3 ± 4.1) compared with fresh samples (76.6 ± 3.3). The percentages of live spermatozoa (P < 0.01) and spermatozoa with intact acrosome (P < 0.05) also decreased significantly in cryopreserved semen (54.0 ± 3.3 and 64.6 ± 5.3) compared with fresh samples (79.3 ± 2.6 and 85.3 ± 1.8). Simultaneously, the total morphological abnormality (%) was found to be significantly (P < 0.01) higher in cryopreserved samples (15.46 ± 2.68) than in fresh semen (3.85 ± 0.63). A total of three mithun cows were inseminated using the cryopreserved semen. All the cows conceived following insemination and gave birth to healthy calves. The study revealed that mithun semen can be cryopreserved efficiently using tris-egg yolk-glycerol diluent, which can be further used for artificial insemination.     

Artificial insemination of sheep: the fertility of semen extended in diluents containing egg yolk and inseminated soon after dilution or stored at 5 degrees C for 24 or 48 hours.

In an experiment involving the artificial insemination (AI) of 1175 ewes, ram semen was diluted 10- or 30-fold in a buffered glucosesaline solution containing either 1.5% or 6% (v/v) egg yolk. Part of each semen collection was used undiluted for control AI of 10⁸ sperm/dose. Diluted samples were reconcentrated to 10⁹ sperm/ml by centrifugation and, from these preparations, 10⁸ spermatozoa were inseminated in a standard volume of 100 μl. Fertility was assessed by 28–45 day non-returns to oestrus.The processes of dilution and reconcentration caused a significant drop in the non-return rate (NRR) and cooling to 5°C and storage for up to 48 hrs at this temperature gave a further large, and highly significant, reduction in NRR. There was no significant effect of level of egg yolk in the diluent on NRR.