Male reproductive biotechnologies in South American Camelids Part II: Semen dilution, cryopreservation and artificial insemination.

Even though South American Camelids (SAC) are a rustic species, adapted to harsh environments, their ability to reproduce is low in their natural habitat. Conception and birth rates in camelids vary from 50 % to 90 %. This depends on the mating system used, sire and dam fertility, postpartum interval, environmental conditions, and nutrition.

Male reproductive biotechnologies in South American Camelids Part I: Semen collection, evaluation and handling.

This review describes the first steps necessary to apply any reproductive biotechnology in South American camelids (SAC) semen or sperm: sample collection, evaluation and handling. In camelids, the length and position adopted for mating and the site of semen deposition have conditioned semen collection methods. The advantages and disadvantages of available collection methods are summarized.

Pre- and Post-Thaw Addition of L-Carnitine and Pyruvate: Effect on Stallion Sperm Parameters.

The addition of antioxidants to cryopreservation media reportedly improves sperm post-thaw quality and reproductive performance after artificial insemination. Therefore, the objectives of this study were to evaluate if the addition of L-carnitine and pyruvate to freezing media, or their addition to samples after thawing, improves the post-thaw quality of equine spermatozoa. Thus, in Experiment 1, stallion semen samples were cryopreserved in: (1) EDTA-glucose-based extender with 20% egg yolk and 5% dimethylformamide (EDTA control); (2) skim milk-based extender with 20% egg yolk and 5% dimethylformamide (milk control); (3) Extender 1 supplemented with 50 mM L-carnitine and 10 mM pyruvate (EDTA-carnitine-pyruvate); and (4) Extender 2 supplemented with 50 mM L-carnitine and 10 mM pyruvate (milk-carnitine-pyruvate).

Evaluation of equine semen frozen in extenders free of egg yolk using two different freezing curves.

A chemically defined cryopreservation extender that maintains seminal parameters is relevant. Fifteen ejaculates from 5 stallions (n= 5; r=3) were diluted in 5 extenders: 1) EDTA-glucose based extender with egg-yolk and dimethylformamide (EY); 2) commercial equine extender (CE); 3) CE with dimethylformamide (CE-3); 4) bovine commercial extender with liposomes (OP); 5) bovine commercial extender with soybean lecithin (BIO), and frozen using a slow and a rapid temperature descent curve. Post-thaw evaluations were: sperm kinematic parameters, viability and acrosome status, membrane lipoperoxidation and DNA fragmentation.

Comparison of different cryoprotectants for freezing donkey (Equus asinus) semen.

The aim of this study was to evaluate two cryoprotectants, dimethylformamide (DMF) and methylformamide (MF) in two concentrations (5 and 7 %) in vitro in donkey semen using a rapid freezing technique and the effect on pregnancy rates in mares. Twenty-four ejaculates from 8 jacks (n = 8; r = 3) were divided into 4 extenders: BotuSemen Gold with 5 % or 7 % MF and 5 % or 7 % DMF, all containing 11 % lactose, 20 % egg-yolk and 0.5 % Equex.

Variations in metabolic parameters of matured porcine oocytes after vitrification-warming.

Background: As the porcine oocyte is the most sensitive to low-temperature damage, it has been difficult to cryopreserve compared to those from other domestic animals. However, at present, vitrification is used as a method for the cryopreservation of both oocytes and embryos in this species.

Aim: Our aim was to analyze alterations in metabolic parameters in vitrified-warmed matured porcine oocytes at different post-warming recuperation times.

Evaluation of DNA integrity and chromatin condensation in cat sperm collected by urethral catheterization and epididymis slicing.

The aim of this study was to evaluate the effectiveness of the original sperm chromatin dispersion (SCD) assay and the toluidine blue (TB) stain to assess DNA fragmentation and chromatin condensation, respectively, in cat sperm obtained by urethral catheterization (CT) and epididymis slicing (EP). CT and EP samples were collected from the same cat, and sperm motility, concentration, morphology, DNA integrity and chromatin condensation were evaluated. As controls, aliquots of the samples were incubated with 0.

Use of commercial extenders, with and without the addition of egg yolk, for cooling llama semen.

The objective of this study was to evaluate the effect of two commercial extenders, AndroMed® (AM) and Androstar® Plus (AS) both with and without the addition of egg-yolk (EY), for cooling llama semen. A total of sixteen ejaculates were collected from four males. Each ejaculate was divided into four aliquots and diluted with: AM, AM with 20 % EY (AM-EY), AS and AS with 20 % EY (AS-EY) and then cooled to 5 °C in an Equitainer®.

Evaluation of DNA fragmentation in dog sperm using the sperm chromatin dispersion test.

The study's objective was to adapt the Sperm Chromatin Dispersion (SCD) protocol to evaluate sperm DNA fragmentation and implement a fragmentation control in dogs. Correlation between DNA status and routine sperm parameters was also analysed. To adapt the SCD, two different mercaptoethanol (ME) concentrations were assayed (2.

Effect of hCG administration on Day 7 post-mating on accessory corpus luteum development and progesterone concentration in llamas.

A high embryo loss rate has been reported in llamas. As strategies that lead to an increase in plasma progesterone (P4) concentration might improve fertility, the aim of the present study was to evaluate if the administration of hCG on Day 7 post-mating is useful to develop an accessory corpus luteum (CL), increasing plasma P4 concentration. Twenty (n = 20) female llamas, ranging between 5 and 10 years of age and four (n = 4) males of proven fertility, ranging between 8 and 10 years of age were included in the study.

Dehydration of llama sperm using different osmolarity media and temperatures for preservation.

The objective of this study was to evaluate effects of dehydration on sperm DNA with the aim of eventually using this method for preserving llama spermatozoa. Two experiments were conducted: 1) sperm preservation at 5 °C for 60 days in different hyperosmotic solutions (500, 800, 1000 and 1200 mOsmol/l) (n = 6, replications = 2) and 2) sperm preservation at 5 and -20 °C for 60 days in the same hyperosmotic solutions, with supplementary antibiotics (n = 6, replications = 2). Sperm motility, membrane functional integrity, viability and morphology were evaluated at 0 and 48 h of the preservation period (Experiment 1) and at 30 min and 24 h (Experiment 2).

Air-Drying Llama Sperm Affects DNA Integrity.

The objective of this study was to evaluate the effects of air-drying preservation on llama sperm DNA. Semen collections were carried out using electroejaculation under general anesthesia. A total of 16 ejaculates were processed from 4 males ( = 4, = 4).

Comparison of Extenders With the Addition of Egg Yolk for Cooling Alpaca Sperm Obtained From Deferent Ducts.

The use of non-commercial and commercial extenders for cooling alpaca sperm has already been reported, the latter showing certain advantages over the first. The Andromed® (AM) extender was created for use in ruminants and has also been tested in ejaculated and epididymal alpaca sperm. According to the manufacturer, this extender does not need the addition of egg yolk (EY); however, it is known that the addition of EY to some extenders improves the preservation of cooled sperm.

Uterine and Corpus Luteum Blood Flow Evaluation Prior to Uterine Flushing in Llama Embryo Donors.

The aim of this study was to assess the uterine blood flow (UBF) and corpus luteum blood flow (CLBF) in llamas 8 days post-mating, using color-Doppler ultrasonography (CDU), to determine the possible relationship between vascularization and the presence of an embryo. Adult females ( = 25) were used to monitor ovarian dynamics by palpation and transrectal ultrasonography until detection of a ≥6 mm growing follicle. Females were randomly assigned to one of two groups: Group I ( = 19), were mated and ovulation was induced by a single dose of buserelin (GnRH analog) that same day (Day 0); and Group II ( = 6), only ovulation was induced (control).

Development of a GnRH-PGF Based Synchronization and Superstimulation Protocol for Fixed-Time Mating in Llama Embryo Donors.

The aim of the present study was to evaluate the application of a GnRH-PGF based synchronization and superstimulation protocol for fixed-time natural mating in llama embryo donors. All females ( = 8) received 8 μg IM of GnRH analog (GnRHa; buserelin) on day 0, regardless of follicular status. After eight days, another GnRHa dose was administered followed by 250 μg IM PGF (cloprostenol).

Incubation of frozen-thawed llama sperm with seminal plasma.

Seminal plasma is intimately connected to sperm physiology and particularly in South American Camelids, has demonstrated to be involved in multiple physiological reproductive events. Different percentages of seminal plasma (0%, 10% and 50%) were added to thawed llama semen samples with the objective of evaluating the interaction with cryopreserved sperm over time (0, 1.5 and 3 hr at 37°C).

Dog sperm DNA: Raw semen evaluation with Toluidine blue stain.

The toluidine blue (TB) stain has been used in different species to evaluate the degree of chromatin condensation. The objectives of this study were as follows: simplify the TB stain to evaluate sperm in canine raw semen, verify the staining patterns for this species using this simplified technique and establish a protocol for using dithiothreitol (DTT) as a positive control for TB staining in dogs. Twenty-one ejaculates were collected from 7 adult male dogs; semen was extended, fixed with ethanol 96° and stained with TB using 2 staining times: 15 and 30 min.

Evaluation of the cryoprotective effect of seminal plasma on llama (Lama glama) spermatozoa.

In South American camelids, sperm survival is low after thawing and poor results are obtained when artificial insemination is performed with cryopreserved semen. The aim of this study was to evaluate the effect of different percentages (10% and 50%) of seminal plasma added prior to the process of cryopreservation and also to evaluate the absence of seminal plasma on llama sperm survival after freezing and thawing. A total of 15 ejaculates from five adult llama males (n = 5; r = 3) were evaluated.

Seminal plasma affects the survival rate and motility pattern of raw llama spermatozoa.

Unlabelled: The objectives of this study were to evaluate the effect over time of different percentages of seminal plasma (SP) on llama sperm characteristics in raw semen and correlate the techniques routinely used to evaluate sperm viability and acrosome status with the Fluorescein Isothiocyanate -Arachis hypogea agglutinin/Propidium Iodide (FITC-PNA/PI). Eighteen ejaculates, obtained from 6 male llamas using electroejaculation, were incubated in 0.1% collagenase in HEPES-TALP (HT), centrifuged and resuspended with SP and HT: 0, 10, 50 and 100% SP.

Production, Preservation, and Transfer of South American Camelid Embryos.

The current review summarizes progress in the field of and production of South American Camelid embryos. Both methods require ovarian superstimulation (with FSH and eCG) to obtain multiple ovulations ( embryo production) or to induce follicle growth for oocyte collection ( embryo production). Moreover, superstimulation entails prior administration of hormones that inhibit follicular growth (progesterone, progestagens, and estrogens).

In vitro production of porcine zygotes using intracytoplasmic injection of vitrified sperm.

The objectives of this study were to evaluate if vitrified porcine spermatozoa are able to maintain their capacity to produce zygotes in vitro using intracytoplasmic sperm injection (ICSI) and to evaluate the zygote development in two in vitro atmospheric conditions: 5% CO and tri-gas. A group of porcine oocytes maturated in vitro were injected with vitrified-warmed sperm (treatment group) and another group, with sperm diluted and conserved at 17°C (control group). To evidence parthenogenetic activation, some oocytes were submitted to a Sham test.

Comparison of two cooling protocols for llama semen: with and without collagenase and seminal plasma in the medium.

Seminal plasma (SP) of South American Camelids could interfere with the interaction of spermatozoa with the extenders; therefore it becomes necessary to improve semen management using enzymatic treatment. Our objective was to compare two cooling protocols for llama semen. Twelve ejaculates were incubated in 0.

Evaluation of the acrosomal status in Lama glama sperm incubated with acrosome reaction inducers.

Unlabelled: The objectives of this study were to evaluate the effect of different acrosome reaction (AR) inducers on viability and acrosomal status in llama spermatozoa, by using the FITC-PNA/PI technique and evaluate if there is a positive correlation between the FITC-PNA/PI and the Coomassie blue (CB) staining techniques. After incubating twenty ejaculates in 0.1% collagenase the centrifuged pellets were resuspended in TALP-BSA medium.