Interbirth interval and duration of pregnancy in hares.

The possibility of inducing superfetation in hares by artificial insemination (AI) was investigated. AI performed on various days during the second half of gestation did not result in new pregnancies, but all ongoing pregnancies were terminated 1-4 days after AI. We suggest that copulation during the last week of pregnancy, common among captive hares, may have a similar effect to that of AI in terminating pregnancies and inducing early deliveries. Intervals between successive deliveries that are shorter than the normal duration of gestation do not necessarily indicate superfetation and this phenomenon may be rare among hares in captivity as well as in the wild.     

GnRH-agonist induction of fertile estrus with either natural mating or artificial insemination, followed by birth of pups in gray wolves (Canis lupus)

Although captive populations of endangered species such as the Mexican gray wolf (Canis lupus baileyi) can benefit from artificial insemination to accomplish genetic exchange, reliable techniques for timing insemination are lacking. We used the generic gray wolf (C. lupus) to test the efficacy of a short-acting GnRH-agonist implant, deslorelin, for inducing estrus. Of five females receiving implants on 17 or 18 January 2003, two mated naturally 10-17 days later, and the others were artificially inseminated using fresh semen, one on day 7 and all three on day 11. Relaxin tests revealed that one artificially inseminated female and both naturally mated females were pregnant on 1 March, and all three gave birth to healthy puppies on 4-6 April. Of the artificially inseminated females, only the one who subsequently conceived and gave birth was judged to be in cytologic estrus at the time of insemination. Two females were treated again with deslorelin on 12 January 2004, followed by collection of fecal samples for hormone analysis. One female, who was housed with a male, copulated on day 17 but did not conceive; the other was not with an adult male. Fecal progestin and estrogen profiles suggested that estrus, but not ovulation, was induced. These results indicated that deslorelin could induce fertile estrus in the gray wolf, although individual response varied. Further investigation is needed to better define and control the interval between implant insertion and ovulation for optimal timing of insemination.     

Comparison of endoscopic-assisted transcervical and laparotomy insemination with frozen-thawed dog semen: A retrospective clinical study

The objective of this retrospective clinical study was to compare pregnancy rates obtained after the use of endoscopic-assisted transcervical catheterization (EIU) or laparotomy (SIU) for insemination of frozen-thawed dog semen. Healthy bitches from various breeds were inseminated with semen from multiple donors processed by different freezing centers. Data from 118 inseminations (78 EIU and 40 SIU) performed between 2009 and 2011 were analyzed. Insemination timing was based on vaginal cytology, serum progesterone concentrations, and vaginoscopy. A ureterorenoscope and a CH-5 Transcervical insemination catheter were used for EIU; 28 of the bitches in this group were inseminated twice with the second insemination less than 12 hours after the first. The numbers of live morphologically normal sperm (LMNS) were determined to characterize insemination doses. Overall, pregnancy rate was greater (P < 0.05) in the EIU group (65%) than in the SIU group (45%). Pregnancy rates were greater (P ≤ 0.06) when more than 100 × 10⁶ LMNS were inseminated regardless of insemination method; the greatest pregnancy rate was observed in the EIU group when this insemination dose was used (38/49; 78%). There was no significant difference in pregnancy rate whether one (69%) or two inseminations (64%) were performed in the EIU group. Complications in the SIU group included anesthetic-induced bradycardia during surgery, significant postsurgery pain, seroma formation over the abdominal incision, and delayed wound healing. No complications were noted during or after insemination in the EIU group. In conclusion, these results support the use of EIU as a noninvasive alternative to laparotomy for insemination of frozen-thawed dog semen. In addition, use of more than 100 × 10⁶ LMNS is also recommended for insemination.     

Artificial insemination in the mouse with special reference to the effect of sperm numbers on the conception rate and litter size (author's transl)

Mature female mice (ICR-JCL), 8 to 12 weeks of age, were artificially inseminated at 8:30-9:30 a. m. on the day of estrus vaginal smear (about 3-7 hr after ovulation) with 3.18 X 10(6), 1.83 X 10(6) and 1.15 X 10(6) sperms from four, two and one cauda epididymidis, respectively, of adult males which were suspended in 50 microliter of a modified Krebs-Ringer-bicarbonate solution and incubated at 37 degrees C was under 5% CO2 in air for an hour. Immediately after insemination, pseudopregnancy induced by an artificial penis and a vaginal tampon. Out of 13 females inseminated with 3.18 X 10(6) sperms, 9 females showed placental signs and 8 of them gave birth to the mean 10.5+/-2.20 (M+/-S.D.) young at term. Four of 13 females having received 1.83 X 10(6) sperms became pregnant giving birth to the mean 4.3+/-2.1 (M+/-S.D.) young at term. On the other hand, 5 out of 6 females failed to become pregnant following insemination with 1.15 X 10(6) sperms, and only one showed a placental sign and gave birth to twelve young at term. It is concluded that the conception rate and litter size are both dependent on the number of sperms inseminated and that more than 3 X 10(6) sperms are necessary to get the conception rate and the litter size comparable to those in natural mating.     

Artificial insemination in the European hare (Lepus europaeus syriacus)

A simple technique for artificial insemination in hares was performed and used routinely in mass breeding of hares in captivity. Sperm was collected directly from the epididymides of males. Ovulation was induced by intravenous injection of human chorionic gonadotropin. Female hares were successfully inseminated throughout the year. Pregnancy occurred in 70% of inseminated females; the range in litter size was 1--3 offspring with a mean of 1.4. It was concluded that artificial insemination greatly simplified the breeding of hares in captivity.     

The concept of superfetation: a critical review on a ‘myth’ in mammalian reproduction

Superfetation is understood as another conception during an already ongoing pregnancy. This implies the existence of young of different developmental stages within the female reproductive tract during certain periods of pregnancy. Nevertheless, a clear definition of the term as well as distinct criteria to identify the occurrence of superfetation in a species is missing. The variable anatomy of mammalian reproductive tracts seems to make the occurrence of superfetation more or less likely but impedes the simple evaluation of whether it is present or not. Additionally, adequate determination methods are missing or are difficult to apply at the right time. Superfetation or rather superfetation‐like pregnancies are reported for numerous species including humans, livestock and rodents. The usual criteria to assume a case of superfetation include the finding of discordantly developed young within the uterus during post mortem or parturition of young after a birth interval shorter than the assumed pregnancy length. Often the occurrence of superfetation is concluded because other explanations of reproductive artifacts are missing. Even severe reproductive pathologies are often confused with superfetation. True superfetation or superfetation as a reproductive strategy may exist in some mammals. In the American mink (Neovison (Mustela) vison) and the European badger (Meles meles) superfetation occurs in combination with embryonic diapause. In the European brown hare (Lepus europaeus), superfetation has long been assumed to exist but evidence is still controversial. Superfetation definitely occurs in certain species of poeciliid and zenarchopterid fish, some of which also exhibit viviparity and maternal care. In mammals, the evolution of such a reproductive mechanism poses many interesting evolutionary, endocrine, microbial and immunological questions that require further investigation. Here we review the scant and at times ancient literature on this poorly understood topic. The different manifestations of superfetation are defined and reliable criteria to detect superfetation are outlined. Also, the differentiation of superfetation into a reproductive strategy or as a disrupted, abnormal reproductive function is discussed. Due to the different discussed functional aspects of superfetation, it is appropriate to establish a more detailed scheme to classify the true natural superfetation cases into superfertilization, superconception and superfetation proper. To date, there is no mammal species known for which superfetation proper in terms of finding discordantly developed fetuses has been conclusively demonstrated to be not only a rare occurence but an evolved reproductive strategy.     

Old grandmothers provide essential care to their young granddaughters in a free-ranging group of Japanese monkeys (<Emphasis Type="Italic">Macaca fuscata</Emphasis>)

This study reports 2 cases in which old grandmothers without dependent offspring provided essential care to their young granddaughters in a free-ranging group of Japanese monkeys (Macaca fuscata). In the first case, a 24-year-old grandmother provided essential care for the survival of her 2-month-old granddaughter for at least 6 days during which the mother had temporarily disappeared from the group for reasons unknown. In the second case, a 14-month-old granddaughter began sucking from her 23-year-old grandmother within 6 weeks after her mother gave birth to a younger sibling. For at least 6 months, the grandmother exhibited various patterns of maternal behavior toward her granddaughter. The behavioral data obtained in this study indicated that old yet healthy females without dependent offspring could contribute to the survival of their young grandchildren.     

The programming of circadian flight-activity in relation to mating and the gonotrophic cycle in the mosquito, Aedes aegypti

ABSTRACT. The circadian flight activity of female Aedes aegypti (L.) changes after insemination and during the course of the gonotrophic cycle. Virgin females attain a high level of spontaneous activity within 3–4 days of adult emergence; in LD 12:12 there are peaks of activity at the beginning and end of the light phase. Inseminated females are less than 20% as active as virgins, but exhibit a similar daily pattern. After blood-feeding, inseminated females become almost totally inactive for about 48h and then show a unimodal pattern of activity which is consistent with oviposition behaviour; after oviposition they revert to the basic inseminated pattern and level of activity. Blood-fed virgin females continue to show a high level of spontaneous activity. It is suggested that insemination has the general effect of raising the threshold for activity and that it is necessary before blood-fed females can switch to the gravid behaviour programme. Blood-fed uninseminated females thus continue with behaviour which will maximize the probability of insemination.     

Birth of pouch young after artificial insemination in the tammar wallaby (Macropus eugenii)

Timing of artificial insemination (AI) in marsupials is critical because fertilization must occur before mucin coats the oocyte during passage through the oviduct. In this study, timing and the site of insemination were examined to develop AI in the tammar wallaby (Macropus eugenii). Birth and postpartum (p.p.) estrus was synchronized in 46 females. Epididymal spermatozoa (n=4) or semen collected by electroejaculation (n=42) were inseminated early (4-21 h p.p.) into the urogenital sinus (n=7), the anterior vaginal culs de sac (n=7), the uterus by transcervical catheter (n=5), or the uterus by injection (intrauterine artificial insemination, IUAI) (n=5). A further 16 females were inseminated late (19-48 h p.p.) by IUAI. All females were monitored for birth. A third group of six females was inseminated late (21-54 h p.p.) by IUAI and 0.4-6.6 h later, sperm had reached the oviduct in all animals. In total, an oocyte to which spermatozoa were attached was recovered and two young were born after IUAI using epididymal (n=1) or electroejaculated (n=2) spermatozoa, but no young resulted from insemination at other sites. Two females were successfully inseminated at 43 and 47 h p.p., later than most other animals, and the third was inseminated much earlier (18 h p.p.) but with highly motile spermatozoa. These young represent the first macropodids born by AI and the first marsupials conceived using epididymal spermatozoa.     

Reproductive biology of captive alpine ibex (Capra i. ibex)

We studied the reproductive biology of alpine ibex bred at the Peter & Paul Wildlife Park, St. Gallen, Switzerland, from 1930 through 1943 and 1968 through 1983. Peter & Paul Wildlife Park had supplied most of the original stock used to reestablish ibex populations in the European Alps after they had become extinct. Ibex females were seasonally polyestrous. Their estrous cycle averaged 20 days, and their gestation period averaged 167 days. Females between 1 and 15 years of age on average produced 0.78 young per year; females between 3 and 13 years of age produced 0.99 young per year. The average age at which captive ibex females gave birth was 8.44 years. Sex ratio at birth was unbiased. More singletons than twins were born, but the proportion of twins born during the years 1968 through 1983 was higher than during the earlier study period. There were no differences in the survival rates of male or female and single or twin offspring.     

Effects of neonatal exposure to diethylstilbestrol on early mouse embryo development in vivo and in vitro

Eight-week-old female mice of the NMRI strain that had been treated neonatally with diethylstilbestrol (DES, 5 micrograms/day for five days) or not (controls) were treated with gonadotropins to induce ovulation and then were artificially inseminated. Ova or young embryos were recovered from the oviducts on the morning after insemination and on Days 2, 3, and 4. In other experiments, ova were obtained from inseminated females on the morning after ovulation and cultured in vitro. In DES-treated females, a few zygotes developed to the 4-cell stage, but no more advanced stages were seen. Under in vitro conditions, zygotes from DES-treated females developed into blastocysts and to the implantation stage, but the incidence of these stages was lower than with zygotes from controls. Our results point to an abnormal oviductal function in DES-treated females that is not compatible with early embryo survival, even though an additional zygote factor contributing to degeneration of early cleavage stages cannot be excluded.     

Pregnancies following long luteal phases in southern white rhinoceros (Ceratotherium simum simum)

All extant species in the Rhinocerotidae family are experiencing escalating threats in the wild, making self-sustaining captive populations essential genetic reservoirs for species survival. Assisted reproductive technologies (ARTs) will become increasingly important for achieving and maintaining ex situ population sustainability and genetic diversity. Previous reports have shown that a large proportion of captive southern white rhinoceros (SWR) females are irregularly cyclic or acyclic, and that cycling females display two different estrous cycle lengths of approximately 30 or 70 days. It has been suggested that the longer estrous cycle length is infertile or subfertile, as no term pregnancies have been observed following long cycles. Here we report the achievement of two pregnancies following long luteal phases, using ovulation induction and artificial insemination with either fresh or frozen-thawed semen. One female SWR conceived on the first insemination attempt and gave birth to a live offspring. A second female conceived twice in consecutive long cycles although the first embryo was resorbed by 33 days post-insemination. A pregnancy from this female's second insemination is ongoing with expected parturition in November 2019. Whether prolonged estrous cycles in SWR are subfertile or infertile in natural breeding situations remains unclear. However, our findings demonstrate that the application of ARTs following prolonged cycles can result the successful establishment of pregnancies in SWR. Therefore, with ARTs, female SWR otherwise considered nonreproductive due to long estrous cycles may still have the potential for representation and contribution to the ex situ population.     

Measurement of EPF for detection of cow pregnancy using rosette inhibition test

Early embryonic death of calves due to sub-fertility in cows is of great economic concern to dairy industry. Early pregnancy factor (EPF) is a secretory protein with pregnancy associated immunosuppressive properties. Rosette inhibition test (RIT) was used to detect EPF in inseminated dairy cows. Blood samples were collected at two intervals, 1-3 and 5-7 days after insemination from 23 inseminated and 18 non-inseminated control cows for RIT and pregnancy diagnosis performed between 42 and 45 days on palpation. The study indicates that RIT (P<0.05) has the potential to distinguish pregnant from non-pregnant dairy cows in the first week of pregnancy.     

Sperm distribution and fertilization after unilateral and bilateral laparoscopic artificial insemination with frozen-thawed goat semen

Generally, laparoscopic artificial insemination (LAI) provides a higher success rate than of cervical insemination in goats. However, the sperm distribution after LAI in goats remains unknown, particularly when frozen-thawed semen is used. This study evaluated the distribution of frozen-thawed goat spermatozoa after LAI and compared the effects of sperm numbers and deposition sites (unilateral and bilateral sites) on pregnancy rate. In experiment 1, the frozen-thawed spermatozoa were stained either with CellTracker Green CMFDA (CT-Green) or CellTracker Red CMPTX (CT-Red), and in vitro evaluations of viability and motility were performed. In experiment 2, the labeled spermatozoa were deposited via LAI into the left (CT-Green) and right (CT-Red) uterine horns (n = 4). After ovariohysterectomy (6 hours after insemination), the distributions of green- and red-colored spermatozoa were assessed via tissue section, flushing, and the oviductal contents were also collected. Experiment 3 was designed to test the pregnancy rates in a group of 120 does after LAI using different numbers of spermatozoa (60 and 120 × 10⁶ sperm per LAI) and different deposition sites. The results demonstrated that the fluorochromes used in this study did not impair sperm motility or viability. Frozen-thawed goat spermatozoa can migrate transuterinally after LAI, as evidenced by the observations of both CT-Green– and CT-Red–labeled spermatozoa in both uterine horns. Lower numbers of spermatozoa (60 × 10⁶) that are inseminated unilaterally (either ipsilateral or contralateral to the site of ovulation) can efficiently be used for LAI in goats (with a 56.67% pregnancy rate).     

Pregnancy in female house mice exposed to urinary chemosignals from other females.

Seven experiments were performed to investigate pregnancy termination, urinary chemosignals, and litter sex ratio variation in female house mice. Experiments tested the effects of urine from adult and prepubertal females, housed individually or in groups, on successful insemination and litter production by females treated at different times and for different periods during the 3 weeks before mating and during gestation. Treatment of females with urine from adult females housed eight per cage or with urine pooled from eight adult females housed individually for 2 or 3 weeks before mating resulted in fewer successful pregnancies and significantly more female-biased litters. Treatment with urine from adult or prepubertal females housed eight per cage or with urine pooled from eight mice housed individually for the first 6 days of gestation or throughout pregnancy resulted in a significant increase in the rate of pregnancy termination. These treatments resulted in lower body weights at birth and slower growth rates in all males and in some females. Puberty was delayed in female progeny from urine-treated dams in five of seven experiments, and these young females attained first oestrus at greater mean body weights than mice in other treatments. These findings indicate that, in mice, at high population density, communication via a urinary chemosignal can alter reproduction in recipient females. Availability of, and competition for, resources such as food would be greater at higher densities, possibly lowering the probability of reproductive success. Pregnancy termination and delays in reproduction and attainment of sexual maturity might lead to greater successful reproduction at a later time.     

Artificial insemination in Callithrix jacchus using fresh or cryopreserved sperm

Assisted reproductive techniques are needed urgently to facilitate the captive breeding of many New World primate species which are endangered in the wild and to assist the effective genetic management of small colonies. A protocol was devised for artificial insemination in the common marmoset, Callithrix jacchus, using ejaculated sperm obtained by vaginal washing after copulation. A double insemination protocol was employed, with the first insemination taking place the day before ovulation was expected to occur and the second 48 h later. All six females inseminated with fresh ejaculated sperm became pregnant, delivering a total of 16 offspring at term. The gestation lengths and litter sizes were not statistically different from those observed in pregnancies following natural mating. The insemination protocol was adapted for use with cryopreserved ejaculated sperm by including an additional insemination on the day of expected ovulation, to take into account differences in the capacitation time of frozen–thawed sperm compared to fresh sperm. Three out of six females inseminated according to this triple insemination schedule, conceived, although one female subsequently resorbed twin foetuses approximately 100 days later. The remaining two pregnant females delivered four babies at term, one singleton and one set of triplets. In the final group, six females were inseminated with low doses of cryopreserved epididymal sperm using the same triple insemination protocol used for frozen–thawed ejaculated sperm. One female conceived, delivering triplets.     

Long-term sperm storage in the Siberian crane (<Emphasis Type="Italic">Grus leucogeranus</Emphasis> pallas): Analysis of paternity and relatedness under artificial insemination

Using ten microsatellite loci, paternity analysis has been conducted for 71 individuals of the Siberian crane (Grus leucogeranus Pallas) obtained under artificial insemination in Oka Crane Breeding Center in 2001–2014. The fathers of 39 chicks were the sires whose sperm was used for insemination directly before fertilized egg laying. Paternity of 23 fertilizations belonged to the sires whose sperm was used in the beginning or middle of insemination cycle. Nine cases of fertilization resulted from natural copulation of artificially inseminated females with their social partners. The terms of sperm storage in the female’s reproductive ducts before fertilization were 0–6 days in the case of paternity of the last sperm donor and 2–15 days in the case of competing sperm by previous donors. Genetic relatedness by microsatellite loci between breeders of the captive Siberian crane population does not prevent fertilization and does not always lead to inbreeding depression.     

Successful embryo transfer in the silver fox (Vulpes vulpes)

Surgical embryo transfer in the silver fox was investigated as part of a larger project concerning the conservation of endangered canine species using modern artificial reproduction techniques with the farmed fox as a model. The animals were chosen on the basis of synchrony in natural oestrus. The timing of ovulation and artificial insemination was determined by measuring electrical resistance in the vagina. Twenty-nine embryos were flushed from eight humanely killed donor females and transferred surgically into the uteri of eight recipients. One recipient female gave birth to two male pups 47 days after the transfer of four expanded blastocysts and one embryo at the 16-cell stage derived from a donor female flushed 10 days after artificial insemination.     

First copulation increases longevity and fecundity of <Emphasis Type="Italic">Histiostoma feroniarum</Emphasis> (Acari: Astigmata: Acaridida) females

I investigated the influence of insemination at different life stages on female fitness in the mite Histiostoma feroniarum. In this species, males guard immature females at the tritonymph stage to inseminate them immediately after the last moulting. Four groups of females were studied (1) females inseminated naturally, i.e. mating occurred immediately after guarding, and then the male was removed (IF/0M), (2) naturally inseminated females, where after insemination the male was replaced by two additional males (IF/2M), (3) virgin females reared without males (VF/0M) and (4) mature, virgin females to which two virgin males were added 3 days after last moulting (VF/2DM). The results show that females inseminated naturally (IF/0M) have higher longevity and fecundity than either virgin (VF/0M) or late-inseminated females (VF/2DM). Furthermore, longevity and fecundity of the former (IF/0M) was also greater than that of females naturally inseminated and subsequently exposed to males (IF/2M). One may suggest that seminal fluids have a positive effect on female fitness. When delayed insemination occurs, such positive effect may not be observed due to a change in features of the sperm access system. Harassment may explain decreased longevity and fecundity of females inseminated naturally compared to females that received additional males.This revised version was published online in May 2005 with a corrected cover date.     

Suckling behaviour in the pipistrelle bat (Pipistrellus pipistrellus)

A sub-colony of 20 pipistrelle bats was studied during late pregnancy and lactation. Seventeen females gave birth to single young, with 16 young surviving to weaning. Free association and flight were permitted, and mother-infant pairs were identified and marked on the day of birth (day 0). Suckling associations were identified daily (n = 195) and females were never found suckling young other than their own. A series of choice experiments using young aged 0–10 days and 15–25 days indicated that females would permit only their own offspring to suckle during both early and late lactation. During early lactation, mothers actively selected their young and encouraged suckling but did not do so during late lactation. Mothers were most often indifferent to approaches by alien young but occasionally aggressively rejected them. Mothers never aggressively rejected their own young but frequently were indifferent to them. Young attempted to suckle from any mother without discrimination.