Comparison of three protocols for superovulation of brown brocket deer (Mazama gouazoubira)

This study aims to evaluate the ovulation rate and the presence of functional corpora lutea after treatment by three different protocols designed to cause superovulation in brown brocket deer. Six female received an intravaginal device containing 0.33 g of progesterone (CIDR®) for 8 days, followed by 0.5 mg injection of estradiol benzoate at the time of insertion and 265 µg of cloprostenol at the time of removal. Afterwards, the hinds were divided into three groups (n = 2): Treatment A received injection of 600 IU eCG on Day 4 after CIDR® insertion; Treatment B received injection of 300 IU eCG at the same time; and Treatment C received injection of 250 IU FSH dissolved in PVP, also on Day 4 post‐insertion. The treatments were crossed over with 44–48 day intervals after CIDR® removal, such that all the deer were submitted to all three treatments. The mean ovulation rate (Treatment A = 3.40 ± 0.68, Treatment B = 1.40 ± 0.24, Treatment C = 0.80 ± 0.49), total ovarian stimulation (Treatment A = 4.80 ± 1.02, Treatment B = 1.80 ± 0.37, Treatment C = 1.40 ± 0.60), and mean CL diameter (Treatment A = 7.33 ± 0.76 mm, Treatment B = 3.94 ± 0.19 mm, Treatment C = 2.18 ± 0.49 mm) in Treatment A were significantly higher than the mean ovulation rates, total ovarian stimulation, and mean CL diameter in Treatments B and C. The mean fecal progesterone metabolites at the luteal phase in Treatment A (6,277.94±2,232.47 ng/g feces) was significantly different from Treatment C (1,374.82±401.77 ng/g feces). Thus, although fertility was not evaluated directly, Treatment A proved capable of induce superovulation in the species Mazama gouazoubira, presenting the greatest mean ovulation rates, with the formation of functional corpora lutea. lutea. Zoo Biol 31:642‐655, 2012. © 2011 Wiley Periodicals, Inc.     

Comparison of two methods of synchronization of estrus in brown brocket deer (Mazama gouazoubira)

This study aimed to establish a protocol for synchronization of estrus in brown brocket deer (Mazama gouazoubira). Two groups of hinds (n = 3) were submitted to two different protocols: Treatment 1 received an intravaginal progesterone (CIDR^®) device for 8 days, followed by 265 μg injection of cloprostenol at the time of removal; and Treatment 2 received two injections of 265 μg of cloprostenol 11 days apart. After 30 days, each group of three hinds received the other treatment. Treatment efficacy was evaluated by reproductive behavior, fecal progestin and estrogen concentration and the observation of CL by laparoscopy 6 days after the end of estrus. All the hinds (100%) had estrous behavior upon the completion of treatment, but a significant difference occurred between the time of onset, 70.5 ± 5.0 h for Treatment 1 and 52.3 ± 5.6 h for Treatment 2. The mean estrus duration time (34.7 ± 4.50 and 37.0 ± 8.11 h), ovulation rates (5/6 and 4/6), mean CL size (4.85 ± 0.74 and 3.21 ± 0.19 mm) and mean fecal progestin concentration at 6 days after the end of estrus (865.53 ± 76.59 and 1073.35 ± 106.82 ng/g feces) were not significantly different between treatments. There was no difference in fecal estrogen concentrations throughout the treatment and the greatest values of the estrogen:progestin ratio coincided with estrous behavior. Although fertility was not evaluated directly, both treatments were effective in synchronizing estrus in the species M. gouazoubira, with the formation of functional corpora lutea.     

Monitoring ovarian cycles and pregnancy in brown brocket deer (Mazama gouazoubira) by measurement of fecal progesterone metabolites

In the present study, pregnancy and the estrous cycle were monitored in captive brown brocket deer (Mazama gouazoubira) by measuring fecal progestagens with a commercial enzyme immunoassay (EIA), along with behavioral data. Fecal samples were collected twice a week during pregnancy and daily during the estrous cycle and post-partum period. It was possible to distinguish between inter-luteal and luteal phases of the estrous cycle. Behavioral estrus corresponded with low concentrations of fecal progestagens. Samples from two consecutive cycles were available from five hinds, and the mean estrous cycle (n=10) was 26.9+/-1.7 d (mean+/-S.E.M.). However, when two extreme cycles (34 and 37 d) were deleted, the mean estrous cycle was 24.7+/-1.2 d. Three animals became pregnant (gestation ranged from 208 to 215 d). After fertile breeding, progestagen concentration in these hinds remained among luteal phase concentrations throughout pregnancy, with the exception of a few peaks. Within 4 d post-partum, two hinds reached interluteal phase values, while one hind maintained luteal concentrations for at least 1 week.     

First live offspring born in superovulated sika deer (Cervus nippon) after embryo vitrification

The rapid growth in sika deer (Cervus nippon) farming and interest in their conservation is an impetus for development of embryo transfer (ET) procedures. However, a paucity of research has prevented widespread application of ET in this species. The objective of the present study was to establish a multiple ovulation and ET procedure with both fresh and vitrified embryos in sika deer. Multiparous weaned hinds (N = 18) were used as embryo donors during the reproductive season of 2008 at a local breeding farm in China. Estrus was synchronized in donors and recipients (N = 38) by inserting a controlled internal drug release for 12 days (insertion = Day 0). Superovulation was induced with a total of 320 mg of NIH-FSH-P1 (Folltropin-V; Bioniche, Belleville, ON, Canada) given as 40 mg im every 12 h from the afternoon of Day 9 to the morning of Day 13. After estrus was detected, donors were artificially inseminated using a transcervical technique. The embryo recovery rate was 76.8% (63/82), including 1.6% (1/63), 77.8% (49/63), and 1.6% (1/63) blastocysts, morula, and eight-cell embryos, respectively. After transfer of fresh and vitrified embryos, pregnancy rates were 85.7% and 61.6% and birth rates were 64.3% and 53.9% (P > 0.05). In conclusion, we developed a satisfactory multiple ovulation and ET procedure in farmed sika deer using vitrified embryos.     

The red brocket deer (Mazama americana) as a new intermediate host of Taenia omissa (Taeniidae)

A total of 13 metacestodes were collected from the lung and parietal pleura from a red brocket deer (Mazama americana) from the Peruvian Amazon. All metacestodes were identified as cysticerci of Taenia omissa by morphological and molecular analyzes. Cytochrome c oxidase subunit 1 sequences from the new isolate T. omissa had more than 96.8% identity with other Peruvian isolates of the species previously sequenced. Lower similarities (93.8–95.8%) were verified between Peruvian and Canadian isolates. This finding adds a new intermediate host for T. omissa and also expands its geographical distribution.     

Reproductive biology of the wild red brocket deer (Mazama americana) female in the Peruvian Amazon

Knowledge of the reproductive biology is critical for the development of management strategies of the species both in captivity and in the wild, and to address conservation concerns regarding the sustainable use of a species. The present report characterizes some aspects of the reproductive biology of the wild red brocket deer inhabiting the North-eastern Peruvian Amazon region, based on the anatomical and histological examination of the female reproductive organs of 89 wild adult females in different reproductive states. The red brocket deer female presented ovarian follicular waves involving the synchronous growth of a cohort of an average 25 follicles but only one follicle generally survived and continued development, reaching maturity at 4mm. Mean ovulation rate was 1.14 and litter size was 1 fetus. Females presented a low rate of reproductive wastage of 14.3% of embryos. Among the 89 adult females studied, 41 (46.1%) were pregnant and 48 (53.9%) were non-pregnant females. In the Northeastern Peruvian Amazon, conceptions occurred year-round in the red brocket deer but there were peaks in the rate of conception. Estimated yearly reproductive production was 0.76-0.82 young per adult female. Most pregnant females in advanced stage of pregnancy had at least one active CL, suggesting the persistence of CL throughout gestation.     

Disease survey of free-ranging grey brocket deer (Mazama gouazoubira) in the Gran Chaco, Bolivia

Samples from 17 free-ranging hunter-killed grey brocket deer (Mazama gouazoubira) in the Gran Chaco, Bolivia, were collected during June-August 1999. All 17 deer appeared to be in good condition at the time of death. Gross necropsies were performed, serum was collected for serologic evaluation of selected infectious disease agents, and feces and ectoparasites were collected for evaluation of internal and external parasites. Serologic tests were positive for antibodies against bovine respiratory syncytial virus and four Leptospira interrogans serovars, with questionable results for epizootic hemorrhagic disease virus serotypes 1 and 2. No antibodies were detected to Anaplasma marginale, Babesia bigemina, Babesia bovis, Babesia odocoilei, bluetongue virus (serotypes 2, 10, 11, 13, and 17), bovine viral diarrhea virus, Brucella abortus, foot-and-mouth disease virus, infectious bovine rhinotracheitis virus, Mycobacterium avium subsp. paratuberculosis, and parainfluenza-3 virus. Sixty-four percent (7/11) of the deer had endoparasites. Amblyomma spp. ticks were found on seven deer, flies of the family Hippoboscidae on six deer, and lice on six deer.     

Reproductive biology of female Amazonian brocket deer in northeastern Peru

The aim of this study was to provide information on the reproductive biology of brocket deer. Hence, we analyzed female reproductive tracts collected by rural hunters from 1991 to 1998 in the Tamshiyacu-Tahuayo Communal Reserve, northeastern Peruvian Amazon. We characterized the basic reproductive biology of brocket deer, analyzed whether the distributions of conceptions and births are aseasonal, and compared their reproductive productivity in two different areas subject to heavy and slight hunting pressures, respectively. We found that: (1) red and gray brocket deer did not differ in ovulation, fertilization, and pregnancy rates; (2) average number of fetuses per birth was 1.2 for red brocket deer and one for gray brocket deer; (3) sex of fetuses suggests a male biased sex ratio for both species; (4) neither species shows reproductive seasonality; and (5) gross productivity does not differ between heavily and slightly hunted areas. Our results indicate that brocket deer exhibit reproductive characteristics similar to their conspecifics in other parts of their native distribution range.     

Low invasive estrous synchronization protocol for wild animals: an example with melengestrol acetate in brown brocket deer (Mazama gouazoubira)

Deer are sensitive to stressful stimuli by handling and their reproductive physiology could be altered by these procedures, making it necessary to develop less invasive protocols for ART. Melengestrol acetate (MGA), a synthetic progestin administered orally, appears as an alternative for estrous synchronization protocols (ESP), such as reported in cattle. Firstly, we compared two MGA doses (0.5 and 1.0 mg/day/animal), which would have suppression effect in estrous behavior (EB). Eight females were randomly and equally distributed in Group 1 (G1) and Group 2 (G2), which received 0.5 and 1.0 mg/day/animal respectively for 15 days (D1 to D15). Two cloprostenol (CP) applications were performed on D0 and D11. Estrus detection (ED) was performed every day. All females from G1 displayed estrus during treatment period, whereas all females from G2 displayed estrus after treatment, suggesting a suppressive effect of 1.0 mg in the EB. Once the suppressive MGA dose (1.0 mg) was defined, we used this dose for assessing ESP. The same eight females received 1.0 mg/animal for eight days (D-8 to D-1), followed by 0.25 mg of estradiol benzoate on D-8 and 265 μg of CP on D0. Feces for fecal progesterone metabolites (FPM) measurement were collected from D0 until seven days after the last day of estrus. Seven females displayed estrus between 12 and 72 h after CP application, which was followed by a significant increase in FPM levels (except female MG6), suggesting the formation of corpus luteum. After ED, females were placed with a fertile male to assess the fertility of the protocol. Pregnancy was confirmed by ultrasound 30 days after mating in 3/6 individuals. Although the low effectiveness of MGA protocol, it should be considered as a promising alternative in deer ESP since this protocol has less stressful effect on the animal during reproductive management when compared to other ESP.     

Identification of the endangered small red brocket deer (Mazama bororo) using noninvasive genetic techniques (Mammalia; Cervidae)

The small red brocket deer Mazama bororo is one of the most endangered deer in the Neotropics. The great morphological similarities with three other sympatric brocket deer species, coupled with the fact that they inhabit densely forested habitats complicate detection and prevent the use of traditional methodologies for accurate identification of species. The ability to determine the presence of this endangered species in an area is crucial for estimating its distribution range, and is critical for establishing conservation management strategies. Here we describe a fast and reliable noninvasive genetic method for species identification of Mazama species from faeces. We designed a primer set that amplifies a short 224-bp fragment of the cytochrome b and demonstrate its effectiveness in successful amplification of DNA isolated from both tissue and faecal samples. This fragment contains a BSTNI/ECORII digestion site that is unique to the endangered M. bororo. The digested polymerase chain reaction products yielded a 160-bp fragment that is clearly visible in a 2% agarose gel. Two other diagnostic sites were identified to differentiate the other three sympatric species, SspI (M. gouazoubira) and AflIII (M. americana, and M. nana).     

Cryptosporidium parvum in brown brocket (Mazama gouazoubira) from Brazil: First report of the subtype IIaA16G3R1 in cervids

This research had as objective to evaluate the occurrence and to characterize genetically the infections by Cryptosporidium in Mazama gouazoubira. By a non-invasive harvest methodology using trained sniffer dogs to locate fecal samples of cervids, 642 fecal samples were obtained from six Brazilian localities. The cervids species responsible for the excretion of each fecal sample were identified by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), using the mitochondrial cytochrome b target gene (cyst b) and the restriction enzymes Sspl, AflIII and BstN. From this identification, 437 fecal samples of M. gouazoubira were selected for research of Cryptosporidium spp. performed through negative staining with malachite green and polymerase chain reaction (nPCR), with the subunit of 18S rRNA gene, followed by sequencing the amplified products. In the samples that were diagnosed the presence of parasite species with zoonotic potential, genotyping was also performed using nPCR with the subunit of GP60 gene. Statistical analysis consisted of the Fisher exact test to verify the association of the presence of the enteroparasite in relation to the presence of cattle in each locality, and the McNemar tests and Kappa correlation coefficient used to compare the results obtained between the two diagnostic techniques. In the fecal samples of M. gouazoubira the occurrences of Cryptosporidium were diagnosed in 1.6% (7/437) and 1.1% (5/437), respectively, through nPCR and microscopy. Cryptosporidium. parvum was diagnosed in 100% (7/7) of the samples submitted to sequencing (18S gene). The IIaA16G3R1 subtype was diagnosed in five of the C. parvum samples submitted to genotyping (GP60 gene). This is the first world report of C. parvum in M. gouazoubira and subtype IIaA16G3R1 in cervids.     

A histological study of cutaneous glands in the brown brocket deer

We examined the histological structure of 8 skin areas thought to contain cutaneous glands of potential importance in scent communication in 16 brown brocket deerMazama gouazoubira Fisher, 1814, using standard histological techniques. Frontal areas and preorbital sacs had scant glandular development. Sebaceous gland development was prominent in vestibular nasal glands and prepucial glands. Apocrine sudoriferous glands and sebaceous glands were well developed in tarsal glands, the caudal skin area and the interdigital glands of front and hind feet. The tail had a unique arrangement of apocrine sudoriferous glands. Anal glands had moderate glandular development, and metatarsal glands were absent. Several of these glandular areas may be important in the chemical communication among brocket deer.     

Characterization of the complete mitochondrial genome and a set of polymorphic microsatellite markers through next-generation sequencing for the brown brocket deer Mazama gouazoubira

The complete mitochondrial genome of the brown brocket deer Mazama gouazoubira and a set of polymorphic microsatellite markers were identified by 454-pyrosequencing. De novo genome assembly recovered 98% of the mitochondrial genome with a mean coverage of 9-fold. The mitogenome consisted of 16,356 base pairs that included 13 protein-coding genes, two ribosomal subunit genes, 22 transfer RNAs and the control region, as found in other deer. The genetic divergence between the mitogenome described here and a previously published report was ∼0.5%, with the control region and ND5 gene showing the highest intraspecific variation. Seven polymorphic loci were characterized using 15 unrelated individuals; there was moderate genetic variation across most loci (mean of 5.6 alleles/locus, mean expected heterozygosity = 0.70), with only one locus deviating significantly from Hardy-Weinberg equilibrium, probably because of null alleles. Marker independence was confirmed with tests for linkage disequilibrium. The genetic variation of the mitogenome and characterization of microsatellite markers will provide useful tools for assessing the phylogeography and population genetic patterns in M. gouazoubira, particularly in the context of habitat fragmentation in South America.     

Olfactory communication and counter-marking in brown brocket deer <Emphasis Type="Italic">Mazama gouazoubira</Emphasis>

Scent marking by defecation and urination in numerous small latrines may be related to resource defence in brown brocket deer (Mazama gouazoubira). Both males and females seem to be territorial, and both contribute to latrines where their ranges overlap. Latrines could thus potentially function as centres of information exchange and intrasexual competition. Counter-marking occurs when animals respond to invaders' marks with a greater number of marks. The objectives of this experimental study were to determine whether brown brocket deer distinguish dung of presumed invaders from their own and whether they counter-mark such faecal deposits. Two samples of dung (from unknown males or females and the experimental animal) were introduced near the latrines of 21 captive deer (13 males and eight females), and we observed their responses, including investigative (sniffing) and marking (urination and defecation) behaviours. Males investigated the introduced dung and their own latrine significantly more when the dung was from an unknown male than when it was their own. Females investigated unknown female dung significantly more than their own. Males counter-marked the introduced dung and their latrine significantly more when the dung was from an unknown male than when it was their own. Males marked with a shorter latency and at a greater frequency than females. Our data indicated that males counter-mark most intensively dung from male ‘intruders’ which may be related to intrasexual competition and resource defence. Females showed a non-significant tendency to counter-mark same sex intruders.     

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

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

Storage of semen and artificial insemination in deer

Methods of collection and freezing of semen of some deer species and aspects of controlled reproduction associated with the use of frozen-thawed semen by artificial insemination (AI) are discussed.     

Fixed-time artificial insemination in red deer (Cervus elaphus) in Argentina

Synchronization of estrous and fixed-time artificial insemination (FTAI) was conducted during the reproductive season of 2008 (March–April) in a local red deer breeding farm in Argentina. Multiparous suckling hinds (n = 38) were artificially inseminated following hormonal treatment (intravaginal sponge containing 100 mg of medroxiprogesterone acetate). At the time of sponge removal (day 12) 250 IU of eCG and 500 μg of PGF2α were given to each hind. The FTAI was performed at 48–55 h after device removal with cryopreserved semen imported from New Zealand. Rectal-transcervical AI method (similar to that in cattle) was performed and semen was deposited within the uterine body (n = 28) or the cervix (n = 10). Pregnancy was diagnosed by means of ultrasonography 44 days after FTAI. The overall pregnancy rate was 36.8% (14/38). Percentage of does that became pregnant with intrauterine seminal deposition was 42.9% (12/28) whereas pregnancy rate in the hinds with intracervical AI was 20% (2/10; P = 0.27).     

Live rhesus offspring by artificial insemination using fresh sperm and cryopreserved sperm

Artificial insemination (AI) and the cryopreservation of sperm with full reproductive capabilities are vital in the armamentarium of infertility clinics and reproductive laboratories. Notwithstanding the fantastic successes with AI and sperm cryopreservation in numerous species, including humans and cattle, these assisted reproductive technologies are less well developed in other species of importance for biomedical research, such as genetically modified mice and nonhuman primates. To that end, AI at high efficiency in the rhesus macaque (Macaca mullata) and the successful cryopreservation of rhesus sperm is presented here, as are the complexities of this primate model due to differences in reproductive tract anatomy and gamete physiology. Cryopreservation had no effect on the ability of sperm to fertilize oocytes in vitro or in vivo. Post-thaw progressive motility was not affected by cryopreservation; however, acrosome integrity was lower for cryopreserved (74.1%) than for fresh sperm (92.7%). Fertilization rates did not differ when fresh (58.1%; n = 32/55) or cryopreserved sperm (63.8%; n = 23/36) were used for in vitro fertilization. Similarly, pregnancy rates did not differ significantly after AI with fresh (57.1%; n = 8/14) or cryopreserved sperm (62.5%; n = 5/8). Seven live rhesus macaques were born following AI with fresh sperm, and three live offspring and two ongoing pregnancies were obtained when cryopreserved sperm were used. Cryopreservation of rhesus sperm as presented here would allow for the cost-effective storage of lineages of nonhuman primates with known genotypes. These results suggest that either national or international centers could be established as repositories to fill the global needs of sperm for nonhuman primate research and to provide the experimental foundation on which to explore and perfect the preservation of sperm from endangered nonhuman primates.