Transcervical artificial insemination in the brown brocket deer (Subulo gouazoubira): A promising method for assisted reproduction in deer

doi:10.21203/rs.3.rs-2595015/v1

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Transcervical artificial insemination in the brown brocket deer (Subulo gouazoubira): A promising method for assisted reproduction in deer

https://doi.org/10.21203/rs.3.rs-2595015/v1

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published 13 Oct, 2023

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The present study aimed to test the efficiency of transcervical artificial insemination techniques with cervical immobilization (TCAI-CI) or cervical traction (TCAI-CT), associated or not with the use of oxytocin (OT) as a protocol for cervical dilation, in the brown brocket deer (Subulo gouazoubira). The study was carried out in a crossover design using four adult females in two replicates with an interval of 60 days. Estrus was synchronized with oral melengestrol acetate (MGA) associated with estradiol benzoate and sodium cloprostenol. TCAI techniques were performed from 18 to 24 hours after estrus onset. All females received either an i.v. Apply 50 IU OT (G-OT, n = 4) or 1 mL saline solution (G-Control, n = 4) 20 minutes before the TCAI procedure. The TCAIs were performed using frozen-thawed semen from the same batch. Behavioral estrus was observed in 100% of females, within the two replicates, with a mean time of onset of 44.0 ± 22.0 h after cloprostenol application. The average length of the vagina was 13.0 ± 1.2 cm. It achieved a 50% (4/8) success of cervical transposition with semen deposition in the uterine.

Regarding inseminations, most of them (87.5%) were performed using the TCAI-CT technique, and the overall conception rate was 50%. Cervical transposition times (< 1 minute) and TCAI procedures (~ 17 minutes) could be considered satisfactory. Thus, the TCAI-CI and TCAI-CT techniques performance was successful when applied in S. gouazoubira females, regardless of the use of OT as a cervical dilation protocol. This procedure is proposed as a method of choice for artificial insemination with greater applicability in different conservation centers, compared to more advanced reproductive biotechniques, and with a favorable impact on the conservation of deer species.

Biological sciences/Biotechnology/Animal biotechnology

Biological sciences/Biotechnology

Health sciences/Endocrinology

Cervical immobilization

cervical traction

oxytocin

Neotropical deer

The environmental impact of unsustainable exploitation of natural resources, as well as other threats, has led to biodiversity reduction and, subsequently, has also increased the rate of extinction faster than the rate of speciation^1,2. In that context, several reproductive biotechnologies appear as part of conservation strategies^3,4. Among these, germplasm banks are positioned as an essential strategy for ex-situ conservation of genetic resources, enabling the preservation of the genetic diversity of wild animals^5,6.

For animals at risk or threatened with extinction, such as some species of deer, one way to guarantee their existence is to increase their reproductive rates via reproduction biotechniques. However, the application of these biotechniques to wild species is still minimal, mainly due to the lack of basic knowledge of the reproductive biology system of most species, such as deer ⁵⁹. In these animals, even in the wild, there is a high degree of structural sperm abnormalities caused by the increase in inbreeding (loss of genetic variability) due to the population decline caused by environmental changes and human interventions ⁵⁹. This picture is also observed in captive animals, caused by the stress of the reduced space, change of environment, and constant coexistence with other individuals⁶⁰.

Another important reproductive biotechnique is artificial insemination (AI), which is widely performed in cervids via laparoscopic access^5–7. However, there are also reports of AI with semen deposition in the vagina without cervical transposition⁸. Laparoscopic methods for AI are reported in several deer species, including Mazama americana⁸, Subulo gouazoubira⁹ (formerly Mazama gouazoubira; Bernegossi et al., in press), Dama dama¹⁰, Cervus Eldi thamin¹¹, Cervus nippon¹¹, Cervus elaphus¹², Elaphurus davidianus¹³, Odocoileus virginianus¹⁴, and Rangifer tarandus⁷.

Although the viability of the intrauterine AI technique by video laparoscopy is satisfactory in deer species, as reported for S. gouazoubira with a conception rate of 50% using frozen-thawed semen⁹, this surgical procedure is invasive. It can cause reproductive sequelae, such as adhesions¹⁵. On the other hand, transcervical AI (TCAI) is a less invasive option and, therefore, more indicated considering animal welfare. However, the biggest obstacle regarding deer TCAI is the anatomy of the cervix, which, similarly to sheep, has a tortuous cervical canal with reduced dimensions^6,16. Despite this, there is one successful report for the cervical canal transposition and semen deposition in the uterine lumen of a Mazama nemorivaga female, giving rise to a healthy offspring¹⁷. Unfortunately, despite this successful report, there have been no reports of other attempts on Neotropical deer.

In sheep, the cervical canal is considered the main obstacle to TCAI techniques performing¹⁸. Thus, to simplify cervical transposition before performing TCAI, hormone protocols based on prostaglandins (E₂ and E₃)¹⁹ and a combination of oxytocin (OT) and estradiol^20,21 have been tested in sheep. In parallel, the development of specific instruments and techniques for AI in sheep and goats has also facilitated obtaining greater efficiency in procedures performed through the transcervical route. In goats, the cervical immobilization technique using the EMBRAPA forceps is effective in ensuring a high rate of cervical transposition during TCAI procedures and for transcervical embryo collection (95% and 100%, respectively)²².

On the other hand, due to the more significant tortuosity of the ovine cervical canal when compared to goats, it was necessary to perform the cervical clamping/traction technique using Pozzi forceps. This was preceded by the cervical dilation protocol composed of a combination of estradiol benzoate, d-cloprostenol, and OT, obtaining high cervical transposition rates (80–100%) during transcervical embryo collection procedures^23–26. Equivalent cervical transposition success in transcervical embryo collection was achieved by reducing the estradiol benzoate dose (1.0 vs. 0.5 mg) or eliminating its use in sheep with synchronized estrus, maintaining the association with d-cloprostenol and OT²⁷.

Among the Neotropical deer species, brown brocket deer (S. gouazoubira) is one of the most abundant species, both in nature and in captivity. It also has a wide distribution in South America and is classified as “Least Concern” by the IUCN²⁸. Thus, it has been considered an excellent experimental model for threatened deer species (10 of the 17 neotropical deer species)²⁹. The present study aimed to test the efficiency of two TCAI techniques, either with cervical immobilization or cervical traction, associated or not with the use of OT as a protocol for cervical dilation in S. gouazoubira females.

General experimental condition

The Ethics Committee on Animal Use (CEUA) of the School of Agricultural and Veterinarian Sciences from São Paulo State University (Unesp), Jaboticabal, São Paulo, Brazil, approved all animal procedures (protocol number 015312/19) and all methods were performed in accordance with the relevant guidelines and regulations by including a statement in the methods section to this study. We declare that the manuscript complies with the ARRIVE guidelines. This is to the ethical principles adopted by the National Council for the Control of Animal Experimentation (CONCEA). The study was carried out in a crossover design between October 2020 and January 2021, with intervals of 60 days between the end of the first treatment and the beginning of the second so that all females belonged to both experimental groups. Four brown brocket hinds (S. gouazoubira) were kept in individual pens (12m²) with olfactory and sound contact with conspecific females and males, exposed to natural photoperiod fluctuation at the Deer Research and Conservation Center (NUPECCE). Animals were fed with 0.5 kg of pelleted ration diet for horses (Equitech® - Presence® - Paulinia, São Paulo, Brazil) provided once daily in the morning. Approximately 1 kg/deer/day of perennial soybean (Neonotonia wightii), ramie (Boehmeria nivea), or blackberry branches (Morus alba), provided according to their availability in the field. Water was offered ad libitum.

Estrus Synchronization Protocol

Estrus synchronization was based on the hormone protocol described by Tanaka et al. (2020)³⁰. All animals received two daily doses of 0.5 mg of melengestrol acetate (MGA, Premix ®; Pfizer. São Paulo, Brazil) for seven days, considering Day 0 as the start of the estrus synchronization protocol. MGA doses were mixed with mashed banana, an edible fruit for deer. Also, on Day 0, females received an i.m. injection of 0.25 mg of estradiol benzoate (Sincrodiol®; Ourofino Saúde Animal Ltda., Cravinhos, Brazil). Concomitantly with the last dose of MGA, an i.m. injection of 265µg of cloprostenol sodium (Ciosin®, MSD., São Paulo, Brazil) was applied (Fig. 1). Between Days 7 and 10, females were subjected to estrus detection every four hours, with the aid of a fertile male of the same species. One animal, which was more docile, was evaluated using lordosis response to handlers and vaginal mucus evaluation⁶. The estrus rate ([%, number of females in estrus/number of females synchronized] x 100) and the interval from treatment to estrus onset (behavioral estrus detection after administration of cloprostenol sodium, in hours) were calculated.

Cervical dilation protocol and transcervical artificial insemination (TCAI).

Artificial inseminations were performed between 18 to 24 hours after the estrus detection, considering insemination in the final third of the estrus according to the estrus duration in the species⁹. Twenty minutes before the TCAI procedure, the females received an intravenous administration of 50 IU of OT (Ocitocina forte UCB®, UCB, Jaboticabal, Brazil) (G-OT, n = 4) or 1 mL of saline solution (G-Control, n = 4). These drugs were administered in a double-blind trial so that the professional inseminator would not know to which group the females belonged. The OT dose was determined based on Dias et al. (2020)²⁷.

Females were subjected to chemical restraint by i.m. of 7 mg/kg of ketamine hydrochloride (Cetamin®, Syntec, Santana do Parnaíba, Brazil) and 1 mg/kg of xylazine hydrochloride (Xilazin®, Syntec, Santana do Parnaíba, Brazil). For analgesia, a slow infusion of 2.5 µg/kg of fentanyl citrate (Fentanest®, São Paulo, Brazil) and the application of 0.4 mg/kg of dipyrone/hyoscine mixture (Buscofin Composto®, Agener União, Embu Guaçu, Brazil) were administered during and after the procedure, respectively.

Females were positioned on a procedure table in sternal recumbency, maintaining the pelvis position with the pelvic limbs flexed. Attempts on cervical transposition were performed within TCAI procedures through cervical immobilization (TCAI–CI) and cervical traction (TCAI–CT). The two procedures were conducted sequentially on all females in both treatments. When the uterus was reached, semen deposition was performed.

The techniques presented followed the previously described for TCAI-CI in goats²² and TCAI-CT in sheep²⁴. After surgical table organization (Fig. 3A), external cleaning and antisepsis of the perineal and vulvar region, a sterile gauze soaked with 5 ml of 2% lidocaine (2% Lidocaine Chloridate®, Bravet, Rio de Janeiro, Brazil) (Fig. 3E) was gently placed at the bottom of the vaginal fornix for 2 minutes. Then, a vaginal speculum ., manufactured for the species (17 cm long and with light source attached; (Fig. 2A) (Fig. 3B), with non-spermicidal intimate lubricant (K-Y®, Semina, São Paulo, Brazil), was slowly inserted (Fig. 3C, 3D). The vagina length (cm) was determined by marking the semen applicator with a marker (Fig. 2 Bi, Bii). The cervical presentation was classified according to Kershaw. et al. (2005)¹⁸ and the mucus was evaluated for presence, color, appearance (crystalline, crystalline/striated, striated, striated/caseous, and caseous)²² and quantity (1 – tiny, 3 – abundant). The grade projection of the cervix into the speculum was rated from 1 to 3 (Figure D) (1 – poor projection and 3 – good projection).

In the TCAI-CI technique (Fig. 3F), a costume-made 25 cm long Embrapa forceps (Embrapa forceps for CI and AI in small ruminants, Brasília, Brazil; Fig. 2C)²⁴ was inserted inside or under the cervical os or just pinched ventrally, according to the anatomy of the cervical os of each animal (Fig. 3F). In the TCAI-CT (Fig. 3G), one or two 26 cm long Pozzi forceps were fixed laterally to the cervical os (Fig. 2D) (Fig. 3G)., according to the grade of vaginal distension by the speculum. The CT was carried out until reaching every animal's anatomical limit. The difficulty for clamping and traction was classified from 1 (easy immobilization) to 3 (rigid immobilization). Clamping repetitions were made whenever clamping got loose or the cervix positioning did not allow performing the following technique steps. All recurrences were recorded. The variables related to clamping were determined for the TCAI-CI and TCAI-CT techniques.

Cervical transposition attempts were performed with a semen applicator manufactured for the species (17 cm long and with two mandrel options, a Hegar uterine dilator, and a semen applicator) (Fig. 2 Bi, Bii, Biii) (Fig. 3H). During the cervical transposition attempt, the Hegar uterine dilator (Fig. 2 Bi, Bii) (Fig. 3H) was kept in the applicator (Fig. 2 Biii). The length of the transposed cervix (cm), the number of cervical rings transposed, and the grade of tortuosity of the cervical canal (1 – straight to 3 – great tortuosity) were recorded. The degree of difficulty of cervical transposition (1 – easy to 3 – impossible to reach the uterus), the percentage of females in which cervical transposition was successful (complete transposition), the time for achieving cervical transposition (in minutes, from the semen applicator, settled up in the cervical os until reaching the uterus, or when exceeding 2 minutes off transposition attempts), and duration of the AI procedure (in minutes, since the introduction of the speculum until semen deposition or exceeding the transposition time) were determined for the TCAI-CI and TCAI-CT techniques. The percentage of females inseminated according to the place of semen deposition (vaginal, superficial cervical, deep cervical, and uterine), percentage of occurrence of semen reflux, and conception rate was also calculated.

Insemination was performed with a 0.25 mL straw of frozen/thawed semen from the same male and batch, with a total concentration of 50 x 10⁶ spermatozoa/straw. The semen was diluted in tris-yolk-glycerol extender³¹ and presented motility of 40%, vigor 3, acrosome integrity of 87%, membrane integrity of 95%, and 13% of total defects after thawing. After insemination and removal of instruments, the female had the pelvic region elevated for 2 minutes and received a clitoral massage.

Pregnancy diagnosis.

Between 30 to 35 days after AI, all females underwent pregnancy diagnosis by transrectal ultrasonography with a 10-MHz linear transducer (Sonoscape A5v® Domed, Valinhos, São Paulo, Brazil) (Fig. 3I), and the conception rate was calculated. Pregnant females received an i.m. 265 µg of cloprostenol sodium for the induction of luteolysis and embryonic loss to allow the realization of the second replica with the same animals.

Statistical analysis

All results are expressed as mean ± standard deviation or percentage. Due to the low number of available individuals, a common issue within wild animal research, only descriptive data analysis was performed.

All females in the two replicates showed estrus behavior 44.0 ± 22.0 hours after the end of the estrus synchronization protocol (cloprostenol administration). The mean vagina length was 13.0 ± 1.2 cm, and 75% (3/4) of the females showed a papillary cervix os, while 25% (1/4) showed a duckbill cervix os. The presence of translucent mucus was observed 18–24 hours after estrus detection in 100% (4/4, G-control) and 75% (3/4, G-OT) of the females, with a 100% crystalline/striated appearance in females of the G-Control and 50% crystalline and 50% crystalline/striated for females of the G-OT. The mean amount of mucus (1 to 3) was 1.2 ± 1.2.

The results of the efficiency variables of the cervical transposition and TCAI procedures according to the G-Control and G-OT groups are shown in (Table 1.) (Fig. 4) shows the percentages of inseminated females conceived according to the site of semen deposition.

Table 1

Results (mean ± standard deviation) of the efficiency of transcervical artificial insemination techniques¹, by cervical immobilization or cervical traction, in *Subulo gouazoubira* females submitted or not to a protocol of cervical dilatation with oxytocin (G-Control, 1 mL de saline solution; G-OT, 50 IU of Oxytocin. Both were intravenous administered 20 minutes before the AI procedure).
Final points	G-Control	G-OT	Total
Estrus response (%)	100%	100%	100%
Time to estrus (hours after cloprostenol administration)	16 ± 24	16 ± 24	16 ± 24
Degree of cervix projection on the speculum (1–3)*	2.0 ± 1.0	1.5 ± 0.6	1.7 ± 0.3
Degree of difficulty of clamping in the cervical traction technique (1–3)*	1.0 ± 0.0	1.2 ± 0.5	1.1 ± 0.1
Number of repetitions of clamping in the cervical immobilization technique	5.0 ± 3.6	5.0 ± 2.4	5.0 ± 0.0
Number of repetitions of clamping in the cervical traction technique	3.3 ± 2.3	4.0 ± 1.4	3.6 ± 0.5
Length of the transposed cervix (cm)	5.9 ± 1.7	4.0 ± 2.6	4.9 ± 1.3
Number of transposed cervical ring	3.5 ± 1.7	1.5 ± 1.0	2.5 ± 1.4
Degree of tortuosity of the cervical canal (1–3)*	3.0 ± 1.0	3.0 ± 1.0	3.0 ± 0.0
Degree of difficulty of cervical transposition in the cervical immobilization technique (1–3)*	2.2 ± 0.9	3.0 ± 0.0	2.6 ± 0.5
Degree of difficulty of cervical transposition in the cervical traction technique (1–3)*	2.2 ± 0.9	3.0 ± 0.0	2.6 ± 0.5
Success (%) of cervical transposition in the cervical immobilization technique	25% (1/4)	0% (0/4)	12.5% (1/8)
Success (%) of cervical transposition in the cervical traction technique	25% (1/4)	50% (2/4)	37.5% (3/8)
Time for cervical transposition in the cervical immobilization technique (min)	0.6 ± 0.1	1.1 ± 0.5	0.8 ± 0.3
Time for cervical transposition in the cervical traction technique (min)	0.5 ± 0.4	0.2 ± 0.2	0.3 ± 0.2
Time for TCAI – cervical immobilization procedure (min)	9.4 ± 4.0	9.1 ± 2.5	9.2 ± 0.2
Time for TCAI – cervical traction procedure (min)	17.4 ± 2.5	16.5 ± 4.2	16.9 ± 0.6
Occurrence of semen reflux (%)	50% (2/4)	0% (0/4)	25% (2/8)
Conception rate (%)	50% (2/4)	50% (2/4)	50% (4/8)
Artificial insemination procedures using EMBRAPA forceps (%)	25% (1/4)	0% (0/4)	12.5% (1/8)
Artificial insemination procedures using Pozzi forceps (%)	75% (3/4)	100% (4/4)	87.5% (7/8)
¹TCAI was performed 18 to 24 h after the detection of behavioral estrus. All females were submitted to a Melengestrol Acetate-based estrus synchronization protocol. * 1: easy; 3: hard. ( ): animal ratio.

This is the first TCAI study in females of S. gouazoubira, achieving success on cervical transposition with semen deposition in the uterine body in 50% (4/8) of the procedures. Most inseminations (87.5%) were performed using the TCAI-CT technique, and the overall conception rate was 50%. The performance index of the TCAI-CI and TCAI-CT techniques points out the success of the non-surgical artificial insemination procedures in S. gouazoubira females, regardless of the use of oxytocin as a cervical dilatation protocol.

Due to a high-stress stimulus when handling the animal^7,38,42, which may alter their physiological reproductive parameters⁴³. With this regard, oral synthetic progestagens, such as melengestrol acetate (MGA), are an efficient option for estrus synchronization in deer species^30,44 and domestic ruminants^45–48. In the present study, 100% of females showed behavioral estrus approximately 44 hours after the end of the protocol. Similar success (100%) was previously reported for S. gouazoubira using the same oral progesterone³⁰. Thus, the MGA-based estrus synchronization protocol is indicated for its high efficiency and for improving the welfare of the managed females, reducing stress sources, and offering more excellent safety to animals and technicians.^45–48. In deer, an epidural block is problematic and stressful during the post-procedure period, when the animal may recover from sedation without return pelvic limb movement. Thus, in addition to the chemical restraint protocol traditionally used by NUPECCE¹⁷, the association of a slow infusion of fentanyl citrate (2.5 µg/kg) was performed to mitigate the signs of discomfort during the intraoperative period. After the procedure, the females received dipyrone/hyoscine (0.4mg/kg)²⁴ to prevent post-procedural pain sensitivity, as done in sheep.

The TCAI techniques of the present study have great potential for use in other deer species in different conservation centers (Zoos, Breeding Institutions, and Scientific Breeding Institutions) due to their greater practicality compared to other techniques, such as laparoscopy or laparotomy. Thus, they appear as a practical and replicable alternative for the genetic management of a great variety of captive populations, which are necessary genetic resources for the conservation of free-living populations and so the species^3,17. Moreover, the chosen TCAI techniques showed satisfactory results in brow brocket females. However, it was necessary the development new instruments considering the anatomical particularities of the species. Thus, a speculum with a smaller diameter and greater length and a longer semen applicator, based on TCAI–CI for goats²² and TCAI–CT for sheep²⁴, were manufactured.

Moreover, using the Embrapa forceps ensured the successful application of the two TCAI techniques (TCAI–CI, and TCAI–CT). Regarding the TCAI-CI, the immobilization was performed only with the Embrapa forceps. While in the TCAI-CT, Embrapa forceps were used before the Pozzi forceps were fixed, which enabled traction movements in most procedures, being removed soon after.

Some anatomical characteristics, such as vagina length and presentation of the cervical os, have not been previously described in this species. The vagina length determined for S. gouazoubira in the present study was ~ 13 cm, longer than that reported for sheep (4–7 cm; ⁵⁰). The greater depth of the vagina in the brown brocket animals required the development of specific instruments, such as a vaginal speculum and a semen applicator. The observed services had conformation of papilla and duckbill type and did not limit the performance of cervical clamping procedures, either using the Embrapa forceps or the Pozzi forceps. The length of the transposed cervix (~ 5 cm) might be used as a cervical length indicator for S. gouazoubira females. Nevertheless, the number of cervical rings counted (mean of ~ 2.5) seems to have been underestimated, considering the high degree of difficulty for the cervical transposition.

Cervical mucus was observed in most females (87.5%), confirming that they were in estrus. The mucus appearance is a physiological characteristic evaluated to determine the time of ovulation in small domestic ruminants, this being more accurate in goats²² than in sheep⁵¹. Compared to data from domestic species, the mucus observed in the present study (crystalline and crystalline/striated) is related to initial moments of estrus, preceding ovulation by ~ 40 hours²², and may be an indicator of estrus synchrony among them. Regarding Neotropical deer species, there is a lack of data on the moment ovulation occurs in response to estrus synchronization protocols. However, most AI performed at NUPECCE were between 18 to 24 hours after the estrus onset, which is also considered the ideal time for AI after behavioral estrus detection in sheep/goat artificial insemination is performed 18 to 24 hours after CIDR removal.

The main anatomical limitation found for performing AI procedures in brown brocket animals, aside from the longer vaginal length, was the low grade of vaginal distension. Despite some projection of the cervix towards the interior of the speculum, the outer end of the cervix did not reach the vulvar rim. Therefore, the clamping was carried out while still inside the vaginal canal. These characteristics highlighted the need for specific instruments for the species and well-trained technicians to perform the procedures. According to Fonseca et al. (2011)⁵², obtaining an excellent cervical projection into the speculum is essential for performing transcervical functions. It can be facilitated by adjusting the speculum size for each female. Among the females of the study, no variation in the diameter of the vagina would indicate the use of different speculum sizes. The set of anatomical characteristics that restricted the space for manipulation of the cervix was an evident challenge for the performance of TCAI in S. gouazoubira, demonstrated by the need to repeat the clamping between 3 and 5 times per procedure.

The cervical canal tortuosity in brown brocket females was classified at the maximum level, indicating a high degree of tortuosity. In sheep, the cervix is described as a tubular, narrow, long, and tortuous organ⁵³. Among brown brocket females, different patterns of topographic distribution of the cervical os can be found ²⁴, leading to greater or lower ease of cervical transposition. The CT can lead to greater alignment of the cervical canal compared to a technique that uses only CI²². In the present study, the exclusive use of OT before the procedures suggests an improvement in the percentage of cervical transposition only in the CT technique. Failure of cervical transposition by the CI technique in the G-OT may result from not only the lack of OT effect on dilating the cervical canal but also the lack of cervical alignment, thus making it difficult to perceive the dilation of the cervical rings. The cervical transposition times (< 1 minute) and TCAI procedures (~ 17 minutes, from speculum placement to semen deposition) are satisfactory and encouraging for the dissemination of the use of TCAI techniques performed in the present study in S. gouazoubira females, as well as in other deer species. In sheep, 83.5% of intrauterine semen deposition was recorded in a mean time of 32 seconds for cervical transposition ⁵⁴.

The overall conception rate of females was 50%, regardless of whether they had previously received OT. Similar pregnancy success rates were previously reported after laparoscopic artificial insemination in the same deer species (50%)⁹ and for females of Odocoileus virginianus (45%)⁵⁵ and Dama dama (56%)⁵⁶. Oxytocin did not interfere with the success rates of TCAI techniques, except for the apparent reduction in the occurrence of semen reflux. The occurrence of reflux is possibly influenced by the location of semen deposition (more abundant when the insemination is more superficial), which may also decrease the number of sperm that reach the fertilization site⁵⁷.

In the present study, conception occurred in 100% of females where artificial insemination was superficial cervical and in 50% of females with intrauterine semen deposition (Fig. 4). In addition to the satisfactory results on cervical transposition and intrauterine semen deposition, the duration of the procedure and conception rate support the indication of these techniques in S. gouazoubira females, regardless of the use of oxytocin as a promoter of cervical dilatation. The use of minimally invasive procedures, such as those applied in the present study, is widely demanded today, mainly in societies concerned with animal welfare issues ⁵⁸.

Artificial insemination techniques with cervical immobilization or traction were successfully applied to S. gouazoubira females. Most inseminations were performed using the TCAI-CT method (87.5%). The exclusive use of oxytocin as a promoter of cervical dilatation does not seem to have increased the success rates of the techniques, suggesting that its use is unnecessary for Subulo gouazoubira females subjected to TCAI procedures.

Declaration of data availability in the manuscript.

Datasets used and/or analyzed during the current study made available by the author correspond upon reasonable request.

Thanks for the attention

Author of correspondence: [email protected]

Frankham, R., Ballou, J. D., Briscoe, D. A. & McInnes, K. H. A Primer of Conservation Genetics. Journal of Bacteriology (Cambridge University Press, 2004). doi:10.1017/CBO9780511817359.
WWF. Living Planet Report 2020: Bending the Curve of Biodiversity Loss. in (eds. Almond, R. E. A., Grooten, M. & Peterson T.) 83 (WWF, 2020).
Comizzoli, P. Biotechnologies for wildlife fertility preservation. Animal Frontiers 5, 73–78 (2015).
Duarte, J. M. B. Coleta, conservação e multiplicação de recursos genéticos em animais silvestres: o exemplo dos cervídeos. Agrociência 541–544 (2005).
Comizzoli, P. Integrating fertility preservation and cryo-banking into the conservation of rare and endangered deer species. Animal Production Science 60, 1227 (2020).
Rola, L. D. et al. Assisted Reproductive Technology in Neotropical Deer: A Model Approach to Preserving Genetic Diversity. Animals (Basel) 11, 1961 (2021).
Morrow, C. J., Penfold, L. M. & Wolfe, B. A. Artificial insemination in deer and non-domestic bovids. Theriogenology 71, 149–165 (2009).
Zanetti, E. S., Polegato, B. F. & Duarte, J. M. B. Primeiro relato de inseminação artificial com tempo fixo em Veado-mateiro (Mazama americana). in Palestras apresentadas no XVIII Congresso Brasileiro de Reprodução Animal 14 (2009).
Peroni, E. de F. C. Inseminação artificial intrauterina por videolaparoscopia em veado catingueiro (Mazama gouazoubira) com sêmen congelado. (Universidade Estadual Paulista, 2013).
Asher, G. W. et al. Intrauterine insemination of farmed fallow deer (Dama dama) with frozen-thawed semen via laparoscopy. Theriogenology 34, 569–77 (1990).
Locatelli, Y. et al. Laparoscopic ovum pick-up and in vitro production of sika deer embryos: effect of season and culture conditions. Theriogenology 66, 1334–42 (2006).
Carragher, J. F., Ingram, J. R. & Matthews, L. R. Effects of yarding and handling procedures on stress responses of red deer stags (Cervus elaphus). Applied Animal Behaviour Science 51, 143–158 (1997).
Argo, C. McG., Jabbour, H. N., Goddard, P. J., Webb, R. & Loudon, A. S. I. Superovulation in red deer (Cervus elaphus) and Pere David’s deer (Elaphurus davidianus), and fertilization rates following artificial insemination with Pere David’s deer semen. Journal of Reproduction and Fertility 100, 629–636 (1994).
MacLean, R. A., Mathews, N. E., Grove, D. M., Frank, E. S. & Paul-Murphy, J. Surgical technique for tubal ligation in white-tailed deer (Odocoileus virginianus). J Zoo Wildl Med 37, 354–60 (2006).
Aller, J. F., Fernandez, O. & Sanchez, E. Fixed-time artificial insemination in red deer (Cervus elaphus) in Argentina. Animal Reproduction Science 115, 312–316 (2009).
Duarte, J. M. B. & Garcia, J. M. Assistant Reproduction in Brazilian Cervidae. Revista Brasileira de Reprodução Animal 19, 111–121 (1995).
Oliveira, M. E. F. et al. First, live offspring of Amazonian brown brocket deer (Mazama nemorivaga) born by artificial insemination. European Journal of Wildlife Research 62, 767–770 (2016).
Kershaw, C. M. et al. The anatomy of the sheep cervix and its influence on the transcervical passage of an inseminating pipette into the uterine lumen. Theriogenology 64, 1225–35 (2005).
Gusmão, A. L. et al. Coleta transcervical de embriões em ovinos da raça Dorper no semiárido do nordeste brasileiro. Arquivo Brasileiro de Medicina Veterinária e Zootecnia 61, 313–318 (2009).
Wulster-Radcliffe, M. C., Costine, B. A. & Lewis, G. S. Estradiol-17 beta-oxytocin-induced cervical dilation in sheep: application to transcervical embryo transfer. J Anim Sci 77, 2587–93 (1999).
Masoudi, R., Kohram, H., Shahne, A. Z. & Davoud, S. D. M. A. Effect of estradiol and oxytocin on ovine cervical relaxation. AFRICAN JOURNAL OF BIOTECHNOLOGY 11, 2803–2806 (2012).
Fonseca, J. F. et al. Reproductive features and use of an anti-inflammatory drug in estrus-induced dairy goats artificially inseminated in a standing position with cervix immobilization. Reprod Biol 17, 268–273 (2017).
Arrais, A. M. et al. NonSurgical Embryo Recovery from Estrus-Synchronized or Superovulated Morada Nova Ewes: A Feasible Strategy for Sheep Embryo Banking. Biopreserv Biobank 19, 360–368 (2021).
Fonseca, J. F. et al. Cervical penetration rates and efficiency of non-surgical embryo recovery in estrous-synchronized Santa Inês ewes after administration of estradiol ester (benzoate or cypionate) in combination with d-cloprostenol and oxytocin. Anim Reprod Sci 203, 25–32 (2019).
Oliveira, M. E. F. et al. Repeated trans-cervical embryo recoveries in Santa inês ewes subjected to short- or long-term superovulation treatment regimens. Animal Reproduction Science 217, 106469 (2020).
Figueira, L. M. et al. In vivo embryo production, and recovery in lacune ewes after imposing a superovulation treatment regimen is related to the pFSH dose. Anim Reprod Sci 223, 106625 (2020).
Dias, J. H. et al. Successful transcervical uterine flushing can be performed without or reduced dose of oestradiol benzoate in cervical relaxation protocol in Dorper ewes. Reproduction in Domestic Animals 55, 844–850 (2020).
Black-Decima, P. A. & Vogliotti, A. Mazama gouazoubira. The IUCN Red List of Threatened Species e.T29620A22154584 (2016) doi:10.2305/IUCN.UK.2016-2.RLTS.T29620A22154584.en.
González, S. & Barbanti Duarte, J. M. Speciation, evolutionary history and conservation trends of Neotropical deer. Mastozoología Neotropical 27(SI), 35–46 (2020).
Tanaka, Y., Americano, A. P., Galindo, D. J. & Duarte, J. M. B. Low invasive estrous synchronization protocol for wild animals: an example with melengestrol acetate in brown brocket deer (Mazama gouazoubira). Animal Reproduction 17, e20200526 (2020).
Favoretto, S. M., Zanetti, E. S. & Duarte, J. M. B. Cryopreservation of red brocket deer semen (Mazama americana): comparison between three extenders. J Zoo Wildl Med 43, 820–7 (2012).
Morrow, C. J. et al. Seasonal effects on the efficacy of intravaginal CIDR devices for estrous synchronization of farmed fallow deer (Dama dama). Proceedings of the New Zealand Society of Animal Production 52, 165–169 (1992).
Morrow, C. J., Asher, G. W. & Macmillan, K. L. Oestrous synchronization in farmed fallow deer (Dama dama): effects of season, treatment duration and the male on the efficacy of the intravaginal CIDR device. Animal Reproduction Science 37, 159–174 (1995).
Asher, G. W. et al. Relationship between the onset of oestrus, the preovulatory surge in luteinizing hormone and ovulation following estrous synchronization and superovulation of farmed red deer (Cervus elaphus). J Reprod Fertil 96, 261–73 (1992).
Asher, G. W. et al. Oestrous synchronization, semen collection and artificial insemination of farmed red deer (Cervus elaphus) and fallow deer (Dama dama). Animal Reproduction Science 33, 241–265 (1993).
Hosack, D. A. et al. Stag exposure advances the LH surge and behavioral estrus in Eld’s deer hinds after CIDR device synchronization of estrus. Theriogenology 51, 1333–42 (1999).
Zanetti, E. S., Polegato, B. F. & Duarte, J. M. B. Comparison of two methods of synchronization of estrus in brown brocket deer (Mazama gouazoubira). Anim Reprod Sci 117, 266–274 (2010).
Zanetti, E. S., Munerato, M. S., Cursino, M. S. & Duarte, J. M. B. Comparing two superovulation protocols on ovarian activity and fecal glucocorticoid levels in the brown brocket deer (Mazama gouazoubira). Reprod Biol Endocrinol 12, 24 (2014).
Cursino, M. S., Salviano, M. B., Abril, V. v., Zanetti, E. S. & Duarte, J. M. B. The role of chromosome variation in the speciation of the red brocket deer complex: the study of reproductive isolation in females. BMC Evol Biol 14, 40 (2014).
Galindo, D. J., Garcia, J. M., Oliveira, M. E. F. & Duarte, J. M. B. Superovulation with equine chorionic gonadotropin in Marsh deer (Blastocerus dichotomus). SPERMOVA 5, 163–167 (2015).
Zanetti, E. S. & Duarte, J. M. B. Comparison of three protocols for superovulation of brown brocket deer (Mazama gouazoubira). Zoo Biol 31, 642–55 (2012).
Dobson, H. & Smith, R. F. What is stress, and how does it affect reproduction? Animal Reproduction Science 60–61, 743–752 (2000).
Monfort, S. L. et al. Monitoring ovarian function and pregnancy in Eld's deer (Cervus eldi thiamin) by evaluating urinary steroid metabolite excretion. J Reprod Fertil 88, 271–81 (1990).
Quadros, A. P. N. de, Tanaka, Y. & Duarte, J. M. B. Avaliação do acetato de melengestrol (MGA) na sincronização do estro em fêmeas de veado-mão-curta (Mazama nana) e veado-roxo (Mazama nemorivaga): estudo piloto. Revista Brasileira de Reprodução Animal 44, 78–80 (2020).
Patterson, D. J., Kiracofe, G. H., Stevenson, J. S. & Corah, L. R. Control of the Bovine Estrous Cycle with Melengestrol Acetate (MGA): A Review. Journal of Animal Science 67, 1895 (1989).
Safranski, T. J., Lamberson, W. R. & Keisler, D. H. Use melengestrol acetate and gonadotropins to induce fertile estrus in seasonally anestrous ewes. Journal of Animal Science 70, 2935–2941 (1992).
Jabbar, G., Umberger, S. H. & Lewis, G. S. Melengestrol acetate and norgestomet for the induction of synchronized estrus in seasonally anovulatory ewes. Journal of Animal Science 72, 3049–3054 (1994).
Powell, M. R., Kaps, M., Lamberson, W. R. & Keisler, D. H. Use of melengestrol acetate-based treatments to induce and synchronize estrus in seasonally anestrous ewes. Journal of Animal Science 74, 2292 (1996).
Figueira, L. M. et al. Preovulatory follicular dynamics, ovulatory response, and embryo yield in Lacaune ewes subjected to synchronous estrus induction protocols and non-surgical embryo recovery. Theriogenology 145, 238–246 (2020).
Bainbridge, D. R. J., Catt, S. L., Evans, G. & Jabbour, H. N. Successful in vitro fertilization of in vivo matured oocytes aspirated laparoscopically from red deer hinds (Cervus elaphus). Theriogenology 51, 891–898 (1999).
Souza-Fabjan, J. M. G. et al. Vaginal cytology and cervical mucus as tools to predict ovulation time in small ruminants. Trop Anim Health Prod 53, 223 (2021).
Fonseca, J. F., Oliveira, M. E. F. & Viana, J. H. M. Uso de procedimentos não cirúrgicos para a produção, recuperação e inovulação de embriões em pequenos ruminantes. Revista Brasileira de Reprodução Animal 35, 113–117 (2011).
B.R. Candappa, I. & Bartlewski, P. M. A Review of Advances in Artificial Insemination (AI) and Embryo Transfer (ET) in Sheep, with the Special Reference to Hormonal Induction of Cervical Dilation and its Implications for Controlled Animal Reproduction and Surgical Techniques. The Open Reproductive Science Journal 3, 162–175 (2011).
Alvares, C. T. G., Romano, C. C., Brandão, F. Z. & Cruz, J. F. da. The technique of artificial insemination by cervical traction and luteal function in Santa Inês ewes after synchronized estrus with cloprostenol or progestagen-eCG. Revista Brasileira de Reprodução Animal 45, 50–57 (2021).
Mellado, M. et al. Factors affecting reproductive performance of white-tailed deer subjected to fixed-time artificial insemination or natural mating. Reprod Fertil Dev 25, 581–6 (2013).
Fennessy, P. F., Mackintosh, C. G. & Shackell, G. H. Artificial insemination of farmed red deer (Cervus elaphus). Animal Science 51, 613–621 (1990).
Azevedo, O. I., Simplício, A. A. & Machado, R. Influência do refluxo da dose inseminante sobre a fertilidade de cabras moxotó inseminadas artificialmente. in 35a Reunião Anual da Sociedade Brasileira de Zootecnia 129–131 (1998).
Petherick, J. C. Animal welfare issues associated with extensive livestock production: The northern Australian beef cattle industry. Applied Animal Behaviour Science 92, 211–234 (2005).59. Holt, W. V.; Brown, J. L.; Comizzoli, P. Reproductive science as an essential component of conservation biology. In: Reproductive Sciences in Animal Conservation. Springer, New York, NY, p. 3–14, 2014.60. ABREU, C. O.; MARTINEZ, A. C., DE MORAES, W., JUVENAL, J. C., MOREIRA, N. Características reprodutivas de veado-bororó-do-sul ou veado-mão-curta (Mazama nana). Pesquisa Veterinária Brasileira, v. 29, n. 12, p. 993–998, 2009
Holt, W. V.; Brown, J. L.; Comizzoli, P. Reproductive science as an essential component of conservation biology. In: Reproductive Sciences in Animal Conservation. Springer, New York, NY, p. 3–14, 2014.
Abreu, C. O.; Martinez, A. C., De Moraes, W., Juvenal, J. C., Moreira, N. Características reprodutivas de veado-bororó-do-sul ou veado-mão-curta (Mazama nana). Pesquisa Veterinária Brasileira, v. 29, n. 12, p. 993–998, 2009

No competing interests reported.

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Transcervical artificial insemination in the brown brocket deer (Subulo gouazoubira): A promising method for assisted reproduction in deer

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