Intravaginal Artificial Insemination Research Articles

Legal position on canine intravaginal artificial insemination.

Legal position on canine intravaginal artificial insemination.

Artificial insemination in a cat: Report of first successful performance resulted with parturition in Turkey

In this case, semen that handled by urethral catheterization was deposited into the deepest point of the vagina of an anesthetized queen. Intravaginal artificial insemination was applied on the 2nd day of onset of the oestrus and only single insemination was performed. Insemination time was determined by behavioural and cytological evaluations. Two healthy kittens were obtained 62 days after insemination. The case has important reports in terms of being the first artificial insemination performance in cats in Turkey, resulted in parturition.

Intravaginal artificial insemination in dorsal recumbency and pregnancy rate in muzaffarnagari ewes

Intravaginal artificial insemination in dorsal recumbency and pregnancy rate in muzaffarnagari ewes

Detection of macrophages in rabbit semen and their relationship with semen quality

We aimed at the evaluating the occurrence of macrophages in rabbit semen and finding possible relationship between macrophage concentration and spermatozoa quality. The concentration of macrophages in semen samples from broiler rabbit males of lines M91 and P91 (n = 30) without overt evidence of genital tract infections was determined using monocyte/macrophage lineage antigen CD14 and flow cytometry. Then the rabbits were assigned into three groups according to the macrophage concentration in semen (MΦ1 group with less than 1 × 106 macrophages/mL, MΦ2 group with 1.5–3.5 × 106 macrophages/mL and MΦ3 group with more than 8 × 106 macrophages/mL). Spermatozoa viability parameters such as occurrence of apoptotic (Yo-Pro-1) and dead/necrotic (propidium iodide) spermatozoa and plasma membrane integrity (PNA-Fluos) were evaluated using flow cytometry. Sperm motility parameters were determined by CASA (Computer Assisted Semen Analysis). Ultrastructural detection of macrophages was performed using transmission electron microscopy. Spermatozoa fertility potential was examined after intravaginal artificial insemination of rabbit doses. Significantly higher proportions of the apoptotic and necrotic spermatozoa and spermatozoa with lower plasma membrane integrity were revealed in the MΦ3 group compared to MΦ1 and MΦ2 groups. The percentage value of total motility and progressive movement was significantly highest in the MΦ1 group, whilst lowest in the MΦ3 group. The conception rate and the kindling rate were significantly decreased in the group with the highest macrophage concentration (MΦ3). Based on our results we can conclude that the abundance of seminal macrophages in the rabbit semen may be closely associated with poor spermatozoa quality.

Influence of Macrophages on the Rooster Spermatozoa Quality.

The goal of this study was to evaluate the occurrence of macrophages in rooster semen and to investigate their impact on the spermatozoa quality. Ross 308 breeder males (n=30) with no evidence of genital tract infections were used to determine the concentration of macrophages using fluorescently conjugated acetylated low-density lipoprotein (AcLDL). Subsequently, the roosters were divided into two groups on the basis of semen macrophage concentration, and semen quality was compared in two heterospermic samples. We applied computer-assisted semen analysis (CASA) system to determine motility parameters. Fluorescence microscopy and flow cytometry were used to evaluate occurrence of apoptotic and dead spermatozoa. Spermatozoa fertility potential was examined after intravaginal artificial insemination of hens. Eighteen roosters (control group) contained 0.2-3% of macrophages within spermatozoa population and ten roosters (macrophage group) had 10-15% of macrophages. Males from macrophage group had lower (p<0.05) motility parameters (total and progressive movement, velocity curved line) and increased concentration of dead spermatozoa detected by flow cytometry and fluorescence microscopy (p<0.001 and p ˂ 0.05, respectively). Differences (p<0.05) between fluorescent microscopy and flow cytometry in results on spermatozoa apoptosis and viability were observed. No significant difference was found between groups in fertility of spermatozoa. In conclusion, the higher presence of macrophages in rooster semen may have a negative effect on some parameters of rooster spermatozoa evaluated invitro. Furthermore, our study suggests that flow cytometry allows more precise examination of spermatozoa viability and apoptosis in a very short time compared with the fluorescent microscopy.

An efficient method of guinea fowl sperm cryopreservation

France is the only country that practices pedigree selection of guinea fowl for meat production. The increasing risk of line extinction for sanitary or breeding failure reasons makes clear the need for an efficient method of reproductive cell cryopreservation in this species. However, an efficient method of guinea fowl sperm freezing in secured packaging is still lacking. The aim of the present study was to develop such a method. Based on results previously obtained in chickens, different cryoprotectants and freezing/thawing processes were tested and then adapted to guinea fowl. Semen quality was measured by semen viability evaluation and then by fertility measured after intravaginal artificial insemination. The best results (70% fertility with frozen-thawed sperm) were obtained by the use of the permeant cryoprotectant agents dimethyl formamide combined with a freezing rate of 30°C/min. The initial insemination frequency also affected the fertility results: 2 consecutive days of inseminations were needed in the first week to ensure enough filling of the utero-vaginal glands of the guinea fowl hen and thus to get successive fertile eggs. Thereafter, a 2-wk insemination frequency was sufficient. This new method, combining biophysical (cryoprotectant agents, freeze/thaw rate) and zootechnical (artificial insemination frequency) features, is the first cryopreservation method successfully developed in secured packaging for guinea fowl sperm. This method is now available for the practice of gene bank conservation and male reproductive management.

Plumping fluid added to storage medium increases twofold the functional life span of fowl spermatozoa in vitro at 4°C

1. The objective of this study was to examine whether addition of plumping fluid (PF) to Lake's solution (LS) for storage of fowl spermatozoa in vitro at 4°C can prolong survival and improve the quality of spermatozoa.2. In experiment 1, aliquots of spermatozoa were stored in vitro in LS alone and LS containing 10%, 25%, 50% and 75% (v:v) PF for 0.5, 24, 48, 72, 96 and 120 h at 4°C. After the end of each storage period, spermatozoa were evaluated for their viability, mobility and penetrability. Viability was determined using SYBR-14 and propidium iodide (PI) staining. Mobility was assessed using an Accudenz assay. Penetrability was assessed using spermatozoa-inner perivitelline layer (IPL) interaction assay.3. In experiment 2, aliquots of spermatozoa were stored in vitro in LS alone and LS containing 25% and 50% (v:v) PF for 0.5, 24, 48 and 72 h at 4°C, and then fertility of the spermatozoa was evaluated using intravaginal artificial insemination (AI) in hens.4. Storage of spermatozoa in LS alone resulted in loss of viability, mobility, penetrability and fertility within 48 h. In contrast, no loss of viability and penetrability was observed for the spermatozoa stored for 48, 96, 72 and 48 h in LS containing 10%, 25%, 50% and 75% (v:v) PF, respectively. In particular, fertilising capacity was not lost for the spermatozoa stored in the presence of 25% or 50% PF in LS for 48 and 24 h, respectively.5. In conclusion, these findings demonstrated that in vitro exposure of fowl spermatozoa to PF during hypothermic storage in LS prolonged spermatozoa survival. A 25% (v:v) level of inclusion of PF in LS may be effective for the improvement of viability, penetrability and fertilising ability of the stored spermatozoa.

Intravaginal Artificial Insemination in Bitches Using Frozen/Thawed Semen after Dilution in Powdered Coconut Water (ACP‐106c)

The aim of this study was to evaluate powdered coconut water extender (ACP-106c; ACP Serviços Tecnológicos Ltda, ACP Biotecnologia, Fortaleza, Ceará, Brazil) as a diluent for freezing dog semen and the fertility after vaginal insemination of semen frozen therein. Ten ejaculates were collected from five dogs, evaluated fresh, diluted in ACP-106c, 10% egg yolk and 6% glycerol, cooled and frozen. In the first phase of the study, straws with frozen semen were thawed and immediately subjected to the same analysis as the fresh semen and, in addition, to Computer-Assisted Semen Analysis (CASA). In phase 2, 10 bitches that had been subjected to natural breeding during a preceding oestrous cycle were vaginally inseminated with thawed semen that had been re-diluted in ACP-106c. After thawing, a mean of 77% sperm motility was obtained through subjective analysis and 77.3% through CASA. Following artificial insemination, a 60% pregnancy rate was observed, resulting in a 50% parturition rate and a mean litter size of 3.4 (SEM 0.6), with 47.1% males and 52.9% females. ACP-106c can be successfully used for freezing canine semen, and vaginal deposition of such semen yields similar pregnancy rates to those reported in other studies.

Favoring the birth of female puppies after artificial insemination using chilled semen diluted with powdered coconut water (ACP-106c)

The objective was to determine the effect of powdered coconut water extender (ACP-106c) on the proportion of female puppies born. Twenty French Bulldog bitches were subjected to natural mating (NM) and, during the subsequent two estrus periods, were bred by intravaginal artificial insemination (AI), using chilled semen (from the same males) diluted in Tris-egg yolk (AI-Tris) or ACP-106c (AI-ACP-106c). Fresh semen was cooled to 5 °C and maintained at that temperature for 6 h, rewarmed (37 °C for 30 s), and used for AI. Pregnancy and whelping rates following NM were both 100% and were both 90.0% following AI with either extender. Litter size (mean ± SD) was 5.4 ±1.1, 4.7 ± 2.0, and 5.1 ± 2.0 (P > 0.05) for NM, AI-Tris, and AI-ACP-106c, respectively. Furthermore, for these groups, the number of female vs. male puppies born were 2.6 ± 0.6 vs. 2.8 ± 1.0, 2.2 ± 1.0 vs. 2.5 ± 1.1, and 3.4 ± 1.6 vs. 1.8 ± 1.2 (P < 0.05 for AI-ACP-106c only). In conclusion, our hypothesis was supported; AI of semen in ACP-106c extender resulted in a significantly higher proportion of female puppies. Furthermore, this extender yielded acceptable litter size and rates of pregnancy and whelping.

Therapeutic Effect of Frequent Injections of GnRH Analogue in a Beagle with Knobbed Acrosome Abnormality of Sperm

A Beagle dog (3 years old) that ejaculated high percentages (mean ± SE: 29.1 ± 1.2%) of sperm with a knobbed acrosome abnormality and a low number of sperm and that also had a low plasma testosterone (T) level was given 10 subcutaneous injections of 1 µg of gonadotropin-releasing hormone analogue (GnRH-A) at 3-day intervals. The plasma T level and number of sperm increased 12-14 weeks after the first injection. Although the percentages of sperm with knobbed acrosome abnormality did not change after the GnRH-A therapy, the number of sperm and percentage of actively motile sperm increased after the therapy, and a bitch gave birth to 5 healthy puppies after intravaginal artificial insemination with fresh semen collected 14 weeks after the first injection.

A Method for Cryopreserving Semen from Yakido Roosters Using N-Methylacetamide as a Cryoprotective Agent

A method of freezing semen using N-methylacetamide (MA) as a cryoprotective agent was applied to Yakido, a chicken breed designated as a “Natural Monument” under the “Law for the Protection of Cultural Property” (Law No. 214, May 30, 1950) in Japan. Semen was collected from Yakido roosters, and pooled semen was diluted 1: 1 using semen diluent containing no cryoprotective agent. After equilibration at 5°C for 30min, diluted semen was further diluted 1: 1 using semen diluent containing MA at a final concentration of 9%. Diluted semen was packaged into 0.5-mL plastic straws and frozen by placing the straws in liquid nitrogen vapor followed by plunging into liquid nitrogen at -196°C. After storage in liquid nitrogen for about one month, frozen semen was thawed in ice water. Intravaginal artificial insemination (AI) was performed immediately after thawing semen without removing the cryoprotective agent. In Fertility Trial 1, AI was performed once a week for four consecutive weeks. Weekly fertility rates using frozen semen were 83.8±3.8%, 77.1±8.8%, 88.6±5.1%, and 77.3±5.7% for weeks 1, 2, 3, and 4, respectively. Hatchability was over 90% throughout the experimental period. In Fertility Trial 2, the duration of fertility was examined after intravaginal insemination for 2 consecutive days. Daily fertility rates using frozen-thawed semen were 90.0%, 90.0%, 91.7%, 88.9%, 100.0%, 100.0%, 100.0%, 55.6%, 60.0%, 25.0%, 30.0%, 30.0%, and 9.1% for days 1 through 13 after AI, respectively. Overall hatchability was 89.5%. These results indicated that freezing chicken spermatozoa using MA as a cryoprotective agent is a practical method of preserving chicken semen collected from genetically valuable chicken breeds.

Effect of Egg Yolk on Cryopreservation of Rhesus Monkey Ejaculated and Epididymal Sperm

Sperm cryopreservation in rhesus monkeys has not been successful when applied in standard, intravaginal artificial insemination; thus, there is a need for substantial improvement in current cryopreservation protocols. The present study was part of our systematic approach to optimize the cryopreservation procedure. Specifically, we tested whether modification of the concentration of egg yolk, the dilution method, and the time delay between ejaculation and adding egg yolk would result in significant improvement of postthaw motility for ejaculated sperm. We also tested the effects of presence and absence of egg yolk on cryopreservation of ejaculated and epididymal sperm. Our findings indicated that the concentration of egg yolk (2%-50%, vol/vol), the dilution method, and the delay (1-5 hours) in addition of egg yolk had no significant effect on postthaw motility of ejaculated rhesus monkey sperm. The presence of egg yolk yielded significantly higher motility after thawing than samples without egg yolk for ejaculated and epididymal sperm. The present study suggests that as long as egg yolk is present in the extender, details such as the amount of egg yolk, as well as when and how to add the egg yolk, have little impact on the ultimate freezing outcomes for ejaculated rhesus monkey sperm. We also discuss the possible mechanism of the protective role of egg yolk in sperm cryopreservation.

Pregnancy in the domestic cat after vaginal or transcervical insemination with fresh and frozen semen

The objective was to compare pregnancy rates in domestic cats using fresh semen for intravaginal artificial insemination (IVI), either at the time of hCG treatment for induction of ovulation, or 28 h later, and to compare pregnancy rates following IVI or transcervical intrauterine insemination (IUI) of frozen-thawed semen. Eighteen queens were inseminated during 39 estrus cycles. Fresh semen with 13.5+/-5.4 x 10(6) sperm (range, 6.8-22 x 10(6)) collected by electroejaculation from four male cats was used in Experiment 1, and cryopreserved semen (20 x 10(6) sperm, with 70+/-5% post-thaw motility) from one male cat was used in Experiment 2. Serum concentrations of estradiol-17beta and progesterone were determined in most queens on the day of AI and again 30-40 days later. Treatment with 100 IU of hCG 3 days after the onset of estrus induced ovulation in 95% of treated queens. Pregnancy rates to IVI with fresh semen at the time of hCG administration versus 28 h later were not different (P=0.58); overall 33% (5/15) of the queens became pregnant. For frozen-thawed semen, AI was consistently done 28h after hCG administration; IUI and IVI resulted in pregnancy rates of 41.7% (5/12), whereas no queen (0/12) became pregnant by IVI (P=0.0083). In conclusion, an acceptable pregnancy rate was obtained with frozen-thawed semen in the domestic cat by non-surgical transcervical IUI; this method might also be useful in other small felids.

A New Device for Intrauterine Artificial Insemination in the Dog

The intrauterine inseminator (IUI) was developed to provide the method of depositing dog semen into the uterine body instead of the vagina. The IUI consists of a vaginal endoscope, a balloon sheath, and injection catheter. When the endoscope is inserted into the vagina and the balloon expanded with air, the cervical os becomes visible so a injection catheter can be inserted through the cervix for deposition of the frozen-thawed semen. The efficacy of the IUI device was compared to intra-vaginal artificial insemination using semen that had been collected and frozen from pooled sperm-rich fraction of ejaculates collected from two Jindo dog donors. Aliquots of semen were extended with a Tris-egg yolk diluent, centrifuged, the seminal plasma removed, the pellet resuspended with the same diluent, and cooled to 5°C over a 2 h period. A Tris-egg yolk-glycerol extender was added at 5°C; after 1 h, semen was loaded into 0.5 ml straws, and straws were frozen in LN vapor for 5 min, and immersed in LN for storage. The final sperm concentration for freezing was approximately 100×10 6 cells/ml. The straws were thawed at 70°C for precisely 6 sec, 1.5 ml Tris-egg yolk buffer at 38°C added, and the 2 ml of thawed semen was used for a single insemination using the IUI device. Each bitch was inseminated at optimal insemination point, which was estimated by vaginal epithelial cells staining and progesterone concentration analysis. Use of the IUI device resulted in 21 of 26 females giving birth to 89 pups (4.2±1.6 pups per litter), while intra-vaginal AI resulted in 6 of 15 females whelping a total of 17 pups (2.8±1.2 pups per litter). We believe the IUI device is easier to use than previously described devices used for intrauterine insemination. In our experience the expansion of the balloon has a calming effect on the bitch that aids the inseminator. These results indicate that the IUI device was able to provide high fertility with 50 million frozen sperm per insemination and two inseminations. (Asian-Aust. J. Anim. Sci. 2003. Vol 16, No. 2 : 180-184)

Ultrastructural observations on in vivo fertilisation in the brushtail possum, Trichosurus vulpecula, following PMSG/LH superovulation and artificial insemination.

Information on the dynamics of gamete interaction in marsupials is very limited and not available for any species from the major Australian Order Diprotodontia which includes most of the more familiar animals such as kangaroos, possums and the koala. This study addressed this deficiency by examining the ultrastructure of in vivo fertilised eggs from common brushtail possums (Trichosurus vulpecula). Females were superovulated by treatment with 15 IU PMSG and then 4 mg porcine LH 3 days later, and inseminations were performed 910-13 h after LH) using epididymal spermatozoa. Between 33 and 39 h after LH injection females were killed, reproductive tracts excised and the oviduct ampulla segment flushed for eggs. Three of the six eggs examined were fertilised as judged by the presence of sperm remnants in the cytoplasm. On the basis of these eggs it was found that sperm penetration left a large hole in the zona pellucida (ZP), suggesting that sperm zona penetration occurs primarily by the enzymatic action of acrosomal enzymes. Sperm lying within the perivitelline space were lacking both an outer acrosomal membrane and the associated acrosomal contents, while both these structures were found on sperm embedded within the mucoid layer, which is consistent with induction of the acrosome reaction by binding to the ZP. Once inside the egg cytoplasm, the sperm head travelled only a short distance before chromatin decondensation occurred. Fertilised eggs showed signs of cytoplasmic activation including cytoskeleton association with apparently dividing mitochondria and prominent rough endoplasmic reticulum. Unfertilised eggs appeared to be undergoing degenerative changes and lacked any evidence of activation. This study was demonstrated that superovulation and laparoscopic intravaginal artificial insemination provide a system through which perifertilisation events in the possum and other monovular Australian marsupials can be examined experimentally.

Artificial insemination with fresh semen in beagle bitches.

The intravaginal artificial insemination was carried out in bitches with fresh semen and the number of sperms and the quantity of semen required for conception was examined. Bitches were inseminated with 1 ml (group 1) or 3 ml (group 2) of semen. Each group was divided into 4 subgroups (A-D) inseminated with semen containing 2.0, 1.0, 0.5 or 0.25×108 viable sperms, respectively. The conception rates in groups A, B, C and D were 7/8 (89%), 5/15 (33%), 6/13 (46%) and 0/8 (0%), respectively, and in naturally mated group, 18/19 (95%). Groups B, C and D were significantly different in the rate of conception from the control and group A. No significant difference was observed among groups in the number of puppies born. In case of intravaginal artificial insemination with fresh semen in bitches, semen should contain 2.0×108 sperms/ml to obtain a similar rate of conception and number of offspring as in naturally breeding bitches.

Preservation of the Fertilizing Capacity of Cock Semen Incubated In Vitro at 41 C

Cock spermatozoa were incubated for 6 and 24 hr at 41 C with minimum essential medium (MEM), tissue culture fluid (TCF) removed from tissue cultured cells and in the presence of tissue cultured cells (TCC). The TCC were obtained from whole 9-day-old chick embryos. Fertility following intravaginal artificial insemination was 75, 77, and 80% for semen incubated at 41 C for 6 hr in MEM, TCF, and TCC, respectively. Fertility of semen incubated for 24 hr was approximately 50% for all three treatment groups. The MEM alone was as beneficial as TCF or TCC in maintaining in vitro sperm survival. These observations provide a method for maintaining in vitro the fertilizing capacity (as evaluated by intravaginal insemination) of large numbers of cock spermatozoa under conditions of temperature comparable to those of the hen's oviduct.

Effect of ovulation and sperm motility on the migration of rabbit spermatozoa to the site of fertilization

Few spermatozoa were present in the ampullae of females 12 h after intravaginal artificial insemination (AI) when there was no ovulation-inducing stimulus. When ovulation was induced, sperm distributions in the female tract 12 h after AI did not differ from those observed 12 h after natural mating. The number of spermatozoa in the oviductal isthmus was similar in all 3 groups as was the percentage of isthmic spermatozoa exhibiting 'activated' motility. When fertile mating was delayed for 8 or 12 h after coitus with a vasectomized male (i.e. 2 h before or after ovulation), spermatozoa were not present in the ampulla 4 h later. The numbers of spermatozoa recovered from the cranial isthmus after delayed matings and 12 h after natural matings did not differ, but after delayed matings the motility of isthmic spermatozoa was non-progressive or poorly progressive and none exhibited 'activated' motility. Flagellar activity of isthmic spermatozoa recovered 4 h after delayed matings and after natural matings was similarly depressed. These observations indicate that sperm ascent to the tubal ampulla in the sustained phase of transport, though enhanced by ovulation, must also depend on changes in flagellar activity and a specific pattern of motility, both of which appear only after spermatozoa have resided for more than 4 h in the female tract.