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Caspases are crucial mediators of programmed cell death (apoptosis). Apoptosis can occur in spermatozoa during spermatogenesis or epididymal transit, as well as in ejaculated spermatozoa. A high proportion of apoptotic sperm would be a poor indicator of the freezability of a raw seminal sample. Alpaca spermatozoa are notoriously difficult to freeze successfully. Therefore, the objectives of this study were to study caspase activation during incubation (37°C) of fresh alpaca spermatozoa, as well as before and after cryopreservation, to gain some insight into the mechanisms behind the vulnerability of alpaca spermatozoa. Eleven sperm samples were incubated for 4 h at 37°C (Study 1), and 23 samples were frozen using an automated system (Study 2). Caspase-3/7 activation was assessed at 0,1,2,3, and 4 h in samples incubated at 37°C (Study 1); and before/after cryopreservation (Study 2) using CellEvent™ Caspase 3/7 Green Detection Reagent and flow cytometry. The proportions of alpaca spermatozoa with caspase-3/7 activated increased (p < 0.05) after 3-4 h of incubation at 37°C; however, caspase activation was similar before and after cryopreservation (36.2 ± 11.2% vs. 36.6 ± 33.7%, p > 0.05). The high standard deviation found after freezing could be explained by the existence of two subpopulations: one subpopulation where caspase-3/7 activation decreased during cryopreservation (from 36.6 ± 9.1% to 1.5 ± 2.2%), and the other subpopulation where caspase-3/7 activation increased after cryopreservation (from 37.7 ± 13.0% to 64.3 ± 16.7%). In conclusion, after 3-4 h of incubation, caspase-3/7 activation increased in fresh alpaca sperm, whereas cryopreservation affects alpaca sperm samples in different ways., (© 2023 Wiley-VCH GmbH. Published by John Wiley & Sons Ltd.)

The present study examined the effects of different photoperiods and melatonin treatment on plasma prolactin concentrations in guanacos, a South American camelid, in captivity. Fourteen adult female guanacos, not gestating or lactating and isolated from males, were studied. The control group was exposed to natural daylight, during short days (N = 7, 10L:14D) and long days (N = 7, 16L:8D). The treatment group (N = 7, 10L:14D) received melatonin implants every 23 days for 6 weeks during long days. Blood samples were taken at intervals of 1 week for 3 weeks, starting the third week of treatment. Prolactin concentrations were measured using competitive ELISA. Plasma concentrations of prolactin in non-lactating female guanacos have seasonal changes, with a higher concentration (p < .001) in short days (3.50 ± 2.24 ng/ml) than long days (1.10 ± 0.91 ng/ml). Melatonin treatment significantly decreases (p < .05) plasma concentrations of prolactin on the 21st day after the treatment. These findings are the first report of an endogenous circannual rhythm of plasma prolactin concentration and the action of melatonin treatment on prolactin secretion in this wild camelid., (© 2020 Wiley-VCH GmbH.)

The objective of this study was to design a protocol to separate spermatozoa from seminal plasma of raw llama semen without prior enzymatic treatment using a single-layer centrifugation with Androcoll-E ™ (AE). Two experiments were performed: (a) samples were divided into three aliquots (1 ml) that were deposited on the top of 4, 5 or 6 ml of AE and were centrifuged at 800g for 20 min and (b) samples were divided into two aliquots (1 ml) that were deposited on the top of 4 ml of AE and were centrifuged at 600g or 1,000g for 20 min. Columns of 5 and 6 ml of AE showed a total sperm motility (TM) significantly lower, while in the 4 ml column, this parameter was not different from the TM of samples before the AE treatment. The percentage of spermatozoa with intact and functional membranes, normal morphology and intact acrosomes, as well as the percentages of sperm with highly condensed chromatin, was conserved (p ˃ .05) in the three column heights and in the two centrifugation speeds evaluated. In conclusion, the different column heights of AE (4, 5 and 6 ml) and the different centrifugation speeds used (600, 800 and 1,000g) allow separating spermatozoa of raw llama semen without enzymatic treatment, preserving the evaluated sperm characteristics. However, of all the studied treatments, centrifugation in the 4 ml column of AE at 800g would be the method of choice to process raw llama semen samples destined for reproductive biotechnologies., (© 2020 Blackwell Verlag GmbH.)

The low fertility and offspring survival indicators in alpacas can be partially due to their particularity seasonal reproduction that reduces the opportunities of the females to become pregnant within a season, with the survival of the offspring concerned by the availability of food and exposure to diseases that depends on the calving date. Optimizing the date of delivery and reducing its variability are shown as eligible criteria that could be used as selection criteria within the genetic improvement programmes in alpacas, the calving date being a much more appropriate trait to measure and optimize fertility unlike of age at first calving and the calving interval, this due to the reproductive seasonality in camelids. For this study, 6,533 birth date records were taken between 2001 and 2018 of Peruvian alpacas, to estimate the genetic parameters. Models assuming heterogeneity in the residuals were fitted besides classical homogeneous models to address, not only the possibility of forwarding or delaying the calving date, but also the trend to have parturitions in similar dates. The heritability and repeatability ranged from 0.07 to 0.20 for a homogeneity model and from 0.08 to 0.23 for a heterogeneity model, and suggest the possibility of advancing or delaying the calving date. It should be taken into account that the gestation length of camelids makes it difficult to adapt many reproductive traits, and trying to centre the calving date could delay it. It was concluded the feasibility to genetically select the calving date, also in the production of camels and dromedaries, which have the same reproductive characteristics as alpacas. This selection can be combined with other traits. The heterogeneity model was shown to provide a better fit., (© 2020 Blackwell Verlag GmbH.)

The aim of this study was to characterize corpus luteum vascularization and its association with plasma progesterone concentration in early stages of pregnancy, when maternal recognition of pregnancy is expected to occur. In all animals, both plasma progesterone concentration and corpus luteum vascularization increased from Day 6 to Day 8 post-mating and afterwards in non-pregnant llamas they started to decrease to reach basal levels around Days 12 to 14 post-mating, while in pregnant animals, both variables remained elevated until the end of the study. A lineal positive relationship between corpus luteum vascularization and plasma progesterone concentration was observed in pregnant (r 2  = .46, p < .0001) and non-pregnant llamas (r 2  = .66, p < .0001). Pregnant animals showed higher plasma progesterone concentration and corpus luteum vascularization than the non-pregnant ones from Day 12 post-mating until the end of the study (p ˂ .05 and p ˂ .01, respectively). These results suggest that maternal recognition of pregnancy should occur before Day 12 post-mating in order to expand luteal lifespan, maintaining corpus luteum vascularization and progesterone production. Also, the assessment of CL vascularization area could be a useful and non-invasive method for early pregnancy diagnosis due to its association with plasma progesterone concentration., (© 2019 Blackwell Verlag GmbH.)

Semen analysis is a key factor when determining the fertility ability in males. South American camelids, and in particular the alpaca, have been studied very little when compared with other farm animals. The aim of this work was to perform the kinematic characterization of alpaca spermatozoa collected directly from the deferent duct by using CASA-Mot (Computer Assisted Semen Analysis for Motility) technology. Samples were obtained every three days throughout the reproductive season during two periods and with a break of seven days in the middle. During both periods, the quality of the sample's motility and kinematics increased over the first two days and then subsequently decreased. This pattern was similar in all animals. It was concluded that the introduction of resting times can be useful to improve sperm quality for artificial insemination purposes in natural conditions., (© 2018 Blackwell Verlag GmbH.)

The aims of the study were twofold: first, the comparison of the pharmacokinetics parameters of two doses of Progesterone BioRelease ® LA, (BioRelease Technologies, Lexington, KY, USA) one of 300 mg and other of 150 mg and their effects on ovarian dynamics in llamas. Based on the results from the first study, the aim of the second study was to evaluate the effect of the doses of 150 mg of progesterone on follicular activity considering the stage of the largest follicle at the beginning of treatment. The results in Study 1 showed that both doses of the formulation induced plasma progesterone concentrations higher than 1 ng/ml during the first 6 days of treatment in all females, progesterone concentrations steadily decline until Day 5 following by a slowly decrease. The total amount of progesterone released during treatment was higher in Group 300 than in Group 150 (p = 0.045). Mean maximum concentrations were 14.9 ± 2.24 and 14.3 ± 2.16 ng/ml for Group A versus Group B (p = 0.58), and they were registered on Day 1.5 ± 0.22 and 1.7 ± 0.34 days, respectively (p = 0.10). None of the animals of Group A showed progesterone concentration below 1 ng/ml during all studied period. The treatment applied in Study 2 was efficient in inhibiting the ovarian follicular dynamics and to start a superestimulatory treatment. The use of progesterone Biorelease ® LA of 150 mg in comparison with the dose of 300 mg could be more effective in the use of synchronization protocols in llamas for AI or prior to the application of an ovarian superstimulatory treatment., (© 2018 Blackwell Verlag GmbH.)

The follicular fluid exerts an effect on the sperm capacitation of several species; however, these effects vary according to species, both in the sperm motility and in the subsequent acrosome reaction. In this study, the effect of alpaca follicular fluid (aFF) on the motility and acrosome reaction of alpaca spermatozoa was observed, using follicular fluid of three follicle sizes: small (<3 mm), medium (3-6 mm) and large (>6 mm), in a concentration of 30%. Sperm motility at the first hour of incubation with aFF of small follicles was 48.0%, with aFF of medium follicles it was 43.33% and with aFF of large follicles, it was 34.53%, while control averaged 26.00%. At the second hour, control achieved an average of 28.13%, treatment with aFF from small follicles showed an average of 46.53%, with aFF from medium follicles it was 40.00% and with aFF from large follicles it was 35.60%. The acrosome reaction after 4 hours of incubation was 30.06% for control, whereas for aFF of small follicles it was 66.3%, with aFF of medium follicles it was 58.86% and for aFF of large follicles, it was 67.63%. In the case of sperm motility, a significant difference is demonstrated for all treatments in relation to the control at the first hour, whereas only the treatments with aFF of small and medium follicles show a significant difference with respect to the control at the second hour. In the case of the acrosome reaction, all treatments with follicular fluid show a significant difference with respect to the control. It was concluded that alpaca follicular fluid favours sperm capacitation and the acrosome reaction in alpaca spermatozoa., (© 2018 Blackwell Verlag GmbH.)

The use of foetal bovine serum (FBS) in cell culture media is quite common. However, little is known about the effect of FBS on sperm. The severe difficulties in alpaca reproduction demand the search of new methods for in vitro reproductive management. In the present study, we use for the first time FBS as a supplement in the culture medium for sperm in alpaca, and the effect of FBS on motility, acrosome reaction and sperm binding to the zona pellucida in this species was evaluated. A concentration of 10% v/v FBS was used. The sperm motility with FBS at the first hour was 32.8% (vs. control = 30.0%), whereas at the second hour sperm motility with FBS was 30.2% (vs. control = 28.8%). The acrosome reaction reached an average of 44.0% for treatment with FBS (vs. control = 30.1%). The sperm-zona pellucida binding assay showed that the samples incubated with FBS had an average of 2.7 bound sperm (vs. control = 1.7). Only a significant difference was observed for sperm motility at the first hour and for the acrosome reaction. It is concluded that FBS favours the capacitation of sperm in alpaca., (© 2018 Blackwell Verlag GmbH.)

The wild South American camelids developed a strategy of seasonal reproduction during spring and summer with singleton birth. The photoperiod is one of the factors that may modulate this seasonality where light would be translated into a hormonal signal. This study evaluated the influence of changes in daily light intensity on melatonin concentration in captive guanacos under a long-day photoperiod (16 hr light/8 hr dark; 33 '28'S). Mean melatonin concentration was 28.3 ± 20.3 pg/ml, with a maximum of 52.14 ± 17.19 pg/ml at 23:30 and minimum of 14.29 ± 6.64 pg/ml at 08:30 (p < .001). There was a negative association between light intensity and melatonin concentration (r = -0.57; p < .001). The results indicate that guanacos respond to variation in daily environmental light with a hormonal response and point to a circannual rhythm as a function of the photoperiod., (© 2017 Blackwell Verlag GmbH.)

The objective of this study was to detect changes in intracellular reactive oxygen species (superoxide anion and hydrogen peroxide) production and lipid peroxidation during cryopreservation of alpaca spermatozoa. Twelve alpaca semen samples were conventionally cryopreserved. Intracellular superoxide anion and hydrogen peroxide were evaluated by fluorescence microscopy using dihydroethidium (DHE)/YO-PRO-1 and dichlorofluorescein diacetate (H2DCFDA)/propidium iodide (PI), respectively. Evaluations were performed during cooling curve at (1) 25°C, (2) 15°C, (3) 5°C/0 min, (4) 5°C/15 min, (5) 5°C/30 min and (6) after freezing/thawing. Evaluation of lipid peroxidation by measuring malondialdehyde (MDA) was performed at 25°C, 5°C/30 min and after thawing. Maximum percentages of total spermatozoa producing superoxide anion and hydrogen peroxide were found at 5°C/30 min (62.8 ± 6.3% and 30.5 ± 5.6%, respectively), and these results were higher (p < .05) than initial (25°C: 10.8 ± 3.8% and 6.8 ± 0.7%, respectively) and after thawing (29.8 ± 9.5% and 7.5 ± 1.8%, respectively) values. However, considering only viable spermatozoa, production of superoxide anion and hydrogen peroxide during overall stabilization at 5°C (>76% and >91%, respectively) and after thawing (74.9 ± 5.0% and 78.9 ± 2.2%, respectively) was higher (p < .05) than initial values at 25°C (38.7 ± 3.1% and 53.6 ± 2.0%, respectively). Lipid peroxidation at 25°C, 5°C/30 min, and post-thawing were 346.5 ± 99.8, 401.1 ± 64.8 and 527.7 ± 142.8 ng/ml MDA, respectively. These results showed that high percentage of viable alpaca spermatozoa produces intracellular reactive species oxygen (ROS) during the cryopreservation process of alpaca semen., (© 2017 Blackwell Verlag GmbH.)

The aim of this study was to compare the effect of the intramuscular administration of 50 μg of gonadorelin acetate versus natural mating, intrauterine infusion (i.u.) of a physiological relevant dose of either raw llama seminal plasma (SP) or purified beta-nerve growth factor from seminal origin (spβ-NGF) on ovulation rate and corpus luteum (CL) development and function in llamas. Females with a follicle (≥8 mm) were assigned to groups: (i) i.m. administration of 50 μg of gonadorelin acetate (GnRH; positive control; n = 4); (ii) single mating (mating; n = 6); (iii) i.u. infusion of 4 ml of llama SP (SP; n = 4); or (iv) i.u. infusion of 10 mg of spβ-NGF contained in 4 ml of PBS (phosphate-buffered saline) (spβ-NGF; n = 6). Ovaries were examined by power Doppler ultrasonography at 0, 1, 3, 6, 12 and 24 hr after treatment to determine preovulatory follicle vascularization area (VA), and additionally every 12 hr until Day 2 (Day of treatment = Day 0) to determine ovulation. Afterwards, ovaries were examined every other day until Day 8 to evaluate CL diameter and VA. Blood samples were collected on Days 0, 2, 4, 6 and 8 to determine plasma progesterone (P4) concentration. Ovulation rate did not differ (p = .7) among groups, but treatment affected (p < .0001) preovulatory follicle VA. Neither treatment administration nor treatment by time interaction affected (p ≥ .4) CL diameter, VA and plasma P4 concentration. Mating tended (p = .08) to increase CL VA when compared to the seminal plasma group by Day 8. Intrauterine administration of seminal plasma or spβ-NGF does not increase CL size and function when compared to i.m. GnRH treatment, suggesting that the administration route of spβ-NGF influences its luteotrophic effect in llamas., (© 2017 Blackwell Verlag GmbH.)

Although computer-assisted systems for sperm morphometry and morphological analysis are important tools in the study of male fertility, their use in extensive systems in alpacas is limited by factors such as the expense of equipment and the high altitudes of the Andean region. The objectives of this study were to evaluate alpaca sperm head morphometry using a nonautomated digital method and determine the frequency of sperm abnormalities based on strict criteria for sperm morphology in fertile male alpacas. Ejaculates (n = 15) from seven alpacas were collected, and sperm smears stained with modified Papanicolaou were processed. For morphometric analysis, 3,000 sperm (200 cells/sample) images were captured at 400× magnification and Quick Photo MICRO 3.0 software was used for manual measurement of basic (sperm head length, width, perimeter and area) and derived variables (ellipticity, shape factor, elongation and regularity). For morphology assessment, smears were observed at 1000× magnification according to WHO and strict criteria. Average morphometric parameters were length 5.48 μm, width 2.99 μm, perimeter 13.62 μm, area 12.43 μm 2 , ellipticity 1.86, shape factor 1.20, elongation 0.29 and regularity 1.05. Significant between-individual and within-individual differences were found in morphometric parameters. Based on morphometric study, sperm heads were classified as elliptical or normal (49%), long (18%), short (2%), pyriform (12%), round (9%), large (6%) and small (4%). Morphological analysis found no additional sperm head defects in 49% of normal sperm obtained by morphometry, although a 4% incidence of neck/mid-piece defects and a 16% incidence of principal-piece defects were found. We conclude that sperm head morphometry assessment in fertile alpacas using a nonautomated digital method is feasible, and that defects in sperm heads constitute the main morphological alteration (>50% of the sperm population), based on WHO and strict criteria., (© 2016 Blackwell Verlag GmbH.)

Plasma progesterone (P 4 ) concentrations and follicular activity after administration of different P 4 doses were evaluated in 33 adult female llamas treated with intravaginal devices. In Study 1, a group of llamas (n = 10) was treated with an intravaginal device (IVD) containing 160 (n = 5) or 780 mg of P 4 (n = 5). Based on the results from the first study, in Study 2, females with follicles at different stages of development were treated with the IVD containing 780 mg of P 4 (n = 21) or remain untreated (control; n = 12) to evaluate the effect of P 4 on follicular activity. In Study 1, the IVD containing 160 mg of P 4 induced follicular turnover in 60% of females while the remaining 40% of llamas developed persistent follicles. Thus, this device controlled follicular activity in llamas, although it promotes the persistence of follicles present at start of treatment. Conversely, in both studies, the IVD containing 780 mg of P 4 suppressed follicular development and hasten the emergence of a new follicular wave in all females regardless of the follicular phase at insertion. Additionally, in Study 2, this device effectively concentrated the appearance of follicles with ovulatory diameter at a definite time after treatment in comparison with control animals. In conclusion, treatment with an IVD containing 780 mg of P 4 would be considered for the control of follicular activity in llamas as it ensures the presence of a young follicle with ovulatory diameter by day 6 after the end of treatment in all females., (© 2016 Blackwell Verlag GmbH.)

This study evaluated ovarian activity in late gestation and post-partum in guanacos in captivity. Follicular dynamics was monitored every second day from 40 days before and other 40 after delivery by transrectal sonography and by plasma steroids concentrations. Seven out of eight (87.5%) of gestating females presented ovarian follicular activity under progesterone levels >3 nmol/l with maximum follicular size of 8.42 ± 0.83 mm from days 23 to 1 before delivery. After delivery, all females have follicular wave development from day 0 to 38, with larger follicular size and longer follicular wave phases and interwave interval when compared with pre-partum data. During post-partum period, there was a close relationship between follicle size and estradiol-17β concentration, with r = 0.69 at the beginning of growth phase and r = 0.86 in association with the largest dominant follicle. Plasma estradiol-17β concentration varied from 11.92 to 198.55 pmol/l. Plasma estrone sulfate, free estrone and progesterone returned to baseline concentrations during peripartal period and remained basal thereafter. The results described follicular activity during late gestation and early post-partum period. These findings provide relevant information to understand physiological changes occurring during this reproductive key period in seasonal breeders with long gestation duration as New and Old World camelids., (© 2014 Blackwell Verlag GmbH.)

Ovarian follicular growth in non-mated llamas occurs in successive waves that generally superimpose their origin on the regression of the preceding wave (overlapping), originating prolonged sexual receptivity in the species. The aim of this study was to perform an ultrasonographic and endocrine characterization of individual and successive waves in non-mated llamas with a special interest on the overlapping phenomenon. Twelve llamas were examined daily by transrectal ultrasonography for at least two consecutive waves. In six females, blood samples were collected daily at the end of each examination. The development of the largest follicle (F) showed a wavelike pattern with a mean duration of 25 days. All waves evaluated were partially overlapped on the preceding wave and emerged at a mean interval of 15.8 ± 0.5 days. This interwave interval determines a mean overlapping degree of 32% of the wave length. Similarly, mean plasma oestradiol-17β (E2 ) concentrations followed a wavelike pattern. However, E2 concentrations started to decline before the structural regression of the F was observed. Mean basal E2 concentrations remain higher than 10.9 ± 0.6 pmol/l. In conclusion, follicular activity in non-mated llamas is characterized by continuous emergence of successive waves that always overlap the preceding wave with variable degrees. E2 production during the follicular wave is shorter in duration than the morphological development of the F. Finally, the overlapping phenomenon maintains increased plasma E2 concentrations persistently and this could explain the prolonged periods of sexual receptivity registered in llamas., (© 2013 Blackwell Verlag GmbH.)

Azoospermia is a common finding in male alpacas which present for infertility. The challenge is to differentiate azoospermia of testicular origin from non-testicular origin. In several species, alkaline phosphatase (AP) concentrations in seminal plasma have been used as a diagnostic marker of contributions of the testis and epididymis to the ejaculate. The purpose of this study was to determine whether AP assay could differentiate testicular from non-testicular azoospermia in male alpacas. An experimental model of bilateral outflow obstruction (pre-scrotal vasectomy) was used in 22 male alpacas, aged 2-9 years. No reproductive history was available. Animals were submitted for electroejaculation (EE) under general anaesthesia and vasectomy performed. Five weeks later, animals were submitted for EE. Vasectomy was not successful in one animal, which was removed from analysis. AP levels were compared in seminal plasma in the pre- and post-vasectomy samples. The mean ± SEM concentration of AP in pre-vasectomy seminal plasma was 504.29 ± 166.45 U/l (range 10-2910); the post-vasectomy levels were 252.48 ± 81.77 U/l (range 0-1640; p = 0.06). In 71.4% of animals, AP levels decreased, varying from 18% to 100% reduction. Results of this study suggest that AP is not produced exclusively by the testis and epididymis in alpacas and that AP assay is not a valid diagnostic test for determination of origin of azoospermia; the gold standard for diagnosis of origin of azoospermia remains testicular biopsy., (© 2013 Blackwell Verlag GmbH.)

Endometrial expression of oestrogen (ERα), progesterone (PR) and oxytocin receptor (OR) and cyclooxygenase-2 (COX-2) was evaluated from the induction of ovulation to luteolysis in llamas. Ovarian activity was daily assessed by ultrasonography in five females. Ovulation was induced immediately after the detection of an ovulatory follicle by a GnRH injection (Day 0). Endometrial samples were obtained by transcervical biopsies from the left and right horns on day 0 and days 4, 8, 10 and 12 post-GnRH. Blood samples were collected daily for progesterone and estradiol-17β determinations by RIA. An immunohistochemical technique was used to study receptors population and COX-2 expression which were then evaluated by two independent observers. The expression of ERα and PR was highest on day 0 in the luminal epithelium and stroma in association with high plasma estradiol-17β concentrations. Thereafter, a decrease in ERα population was registered on day 4 and a new increase of its expression was observed between days 8 and 12 in those cell types. Conversely, PR population was gradually down-regulated until its lowest expression was reached on day 10 post-GnRH in the luminal epithelium. Content of OR was similar throughout the study in all cell types. The expression of COX-2 was highest from day 8 to 12 post-GnRH in the luminal epithelium, in relation to the time of maximal PGF2α release. Both steroid receptors populations and COX-2 expression were similar between horns. Meanwhile, OR expression was higher in the right than in the left uterine horn. In summary, this study showed that the loss of endometrium sensitivity to progesterone by days 8-10 post-induction of ovulation and the concomitant increase of COX-2 expression could play a key role in the mechanism of luteolysis and somehow be related to the short corpus luteum lifespan of llamas., (© 2013 Blackwell Verlag GmbH.)

Flow cytometry has been shown to be an accurate and highly reproducible tool for the analysis of sperm function. The main objective of this study was to assess sperm function parameters in ejaculated alpaca sperm by flow cytometry. Semen samples were collected from six alpaca males and processed for flow cytometric analysis of sperm viability and plasma membrane integrity using SYBR-14⁄PI staining; acrosomal membrane integrity using FITC-conjugated Pisum Sativum Agglutinin⁄PI labelling; mitochondrial membrane potential (Δψm) by staining with JC-1 and DNA Fragmentation Index (DFI) by TUNEL. The results indicate that the mean value for sperm viability was 57 ± 8 %. Spermatozoa with intact acrosome membrane was 87.9 ± 5%, and viable sperm with intact acrosomal membrane was 46.8 ± 9%, high mitochondrial membrane potential (Δψm) was detected in 66.32 ± 9.51% of spermatozoa and mean DFI value was 0.91 ± 0.9%. The DFI was inversely correlated with high Δψm (p = 0.04; r = -0.41) and with plasma membrane integrity (p = 0.01; r = -0.47). To our knowledge, this is the first report of the assessment on the same sample of several parameters of sperm function in ejaculated alpaca sperm by flow cytometry., (© 2012 Blackwell Verlag GmbH.)

The present study intended to develop the hypoosmotic swelling (HOS) test in alpaca for its use in epididymal spermatozoa, to evaluate the membrane functional integrity and determine an appropriate hypoosmotic solution and whether the incubation time of 15 or 60 min is sufficient for the execution of the test. Hypoosmotic solutions (HS) with the following concentrations were used: 50, 100, 150, 200 and 275 mOsm/kg of sodium citrate tribasic dihydrate and d-fructose. Ten microlitres of epididymal sperm sample was mixed in 150 μL of the respective HS and incubated for 15 or 60 min at 38°C. From the proportion of reacted (swollen) spermatozoa, the 150 mOsm/kg HS was the most sensitive (p < 0.05). The exposure times (15 and 60 min) did not have significant differences (p > 0.05) in the proportion of both strong- and total-coiled sperm tails. In conclusion, 150 mOsm/kg HS and 15 min exposure time are optimal to evaluate the plasma membrane functional integrity through the HOS test in alpaca epididymal spermatozoa., (© 2012 Blackwell Verlag GmbH.)

Camelid semen is characterized by a highly viscous, low-volume ejaculate with a low concentration of spermatozoa that exhibit low progressive motility. The viscous seminal plasma is currently the major impediment to the development of assisted reproductive technologies (ARTs) in camelids. To advance ARTs such as sperm cryopreservation and artificial insemination in camelids, it is necessary to identify the cause of the viscosity and gain an understanding of the role of seminal plasma components on sperm function and fertility. Numerous compounds and proteins have been identified as mediators of sperm function and predictors of fertility in other livestock species, and understanding the importance of specific proteins has progressed the success of ARTs in these species. Current knowledge on the components of camelid seminal plasma is outlined, together with the implications of these components for the development of ARTs in camelids. The cause of semen viscosity, as well as proteins that are present in camelid seminal plasma, is described for the first time. Seminal plasma components are compared with those of other species to hypothesize their role in sperm function and fertility., (© 2012 Blackwell Verlag GmbH.)

The aim of this study was to characterize the distribution of oestrogen receptor (ER)α and ERβ as well as both progesterone receptors isoforms progesterone receptor (PR) A and PRB in the luminal and glandular epithelia and stroma of the endometrium during the different phases of the follicular wave in llamas. Six llamas were examined by transrectal ultrasonography, and a transcervical biopsy was obtained when a follicle at the growing, plateau and regressing phase was recorded. Blood samples were collected at the time of biopsy for hormone determinations. An immunohistochemical technique was used to study receptor populations. Total positive area was evaluated in the different cell types by Image Analysis. Mean diameter measurements of the largest follicle were 6.9, 8.5 and 5.1 mm (p < 0.001) and mean plasma oestradiol-17β concentrations were 27.9 ± 3.26; 30.0 ± 2.79 and 24.0 ± 1.78 pmol/l (p = 0.32) during the growing, plateau and regressing phases, respectively. Immunostaining of ERα was higher in the luminal epithelium during the plateau and regressing phases (p < 0.05) than during the growing phase. More positive cells to ERβ were observed in the glandular epithelium of the growing and plateau phases (p < 0.05) than during the regressing phase. A higher percentage of cells positive to PRB was recorded in the luminal and glandular epithelia during the plateau phase (p < 0.05), while the PRA immunostaining was similar among phases. In brief, this study showed an increased population of ERα and PRB in the luminal epithelium, and only of PRB in the glandular epithelium at the time when an ovulatory follicle is present. The physiological importance of these changes in llamas remains to be elucidated., (© 2009 Blackwell Verlag GmbH.)

Artificial insemination (AI) is poorly developed in camelids owing to the difficulty in collecting high quality semen and the highly viscous nature of the semen. Semen collected by artificial vagina (AV) is often of low quality and must be improved before any further development of AI technology can occur. The present study investigated the effects of adding a cervix-like stricture to the AV, presence of females, collecting semen into Androhep, skim-milk or Tris diluents, and catalase supplementation (0, 100, 200 or 600 units/ml) of Tris diluent on alpaca semen quality parameters. The addition of a cervix-like stricture increased mating length (p < 0.05), whilst the presence of females during semen collection did not improve semen quality parameters (p > 0.05). Collection of semen into Tris diluent improved sperm motility (58.0 +/- 11.9%) compared with the control (34.0 +/- 10.8%; p < 0.05), Androhep (33.5 +/- 10.7%) and skim-milk diluents (28.2 +/- 10.4%). Semen viscosity was reduced by collection into Androhep (4.6 +/- 1.7 mm) and skim-milk diluents (3.6 +/- 1.3 mm) compared with Tris diluent (5.7 +/- 2.1 mm) and no collection medium (9.3 +/- 3.5 mm; p < 0.05). Tris diluent supplemented with 100, 200 or 600 units/ml catalase increased semen viscosity (5.0 +/- 3.2 and 4.9 +/- 3.2 mm). Collection of alpaca semen by AV into Tris diluent increased semen quality facilitating further development of AI technology in alpacas.

Sperm reservoirs in South American Camelids would be crucial for successful fertilization. Since ovulation occurs approximately 36 h after mating, the maintenance of the sperm viability in the oviduct waiting for the ovum is a critical reproductive event. Our study aimed at determining whether the isthmus or the utero tubal junction (UTJ) could function as a sperm reservoir in llama by means of in vivo and in vitro experiments. For the in vivo experiments, the oviducts of adult females with a dominant follicle bigger than 7 mm were examined for the presence of sperm at 6, 18, 24, 28 and 35 h after mating. The results using scanning and transmission electron microscopy showed ultrastructural differences between isthmus and UTJ with respect to (1) predominance of secretory cells in the UTJ and ciliated cells in the isthmus epithelium and (2) cytoplasmic bulbous projection of the secretory cells in the UTJ. Sperm adhered by a mucus-like substance were seen only in the UTJ at 6, 18, 24 and 28 h postmating. Lack of sperm adhered to oviductal mucosa was observed around ovulation (35 h). In vitro experiments demonstrated higher ability of UTJ epithelial cell explants with respect to isthmus explants to bind sperm in a co-cultured system. The anatomical features and the presence of a sperm bonding agent in the UTJ together with the in vitro differential binding of sperm to UTJ explants strongly suggest that both may be feasible mechanisms that facilitate sperm storage in this oviductal region in llama.

The aim of the present study was two-fold. Experiment I: evaluate the effect of buserelin on llama's oocyte maturation after exogenous follicular activity suppression, followed by ovarian superstimulation with different doses of equine chorionic gonadotropin (eCG). Experiment II: compare the number of follicles aspirated and the number of cumulus-oocyte complexes (COCs) recovered according to different doses of eCG followed by buserelin. Experiment I consisted in a control group (without buserelin) and a treatment group (with buserelin), both subdivided according to eCG dose administered: A: 500 IU; B: 1000 IU; C: 1500 IU. The treatment group received a single i.v. dose of 8 microg of buserelin when two or more dominant follicles were found at ultrasound evaluation and 20 h later were subjected to surgery. In group A, 83% of the llamas did not respond to superstimulation. In groups B and C differences were observed between the control and the treatment groups for the degree of COCs maturation (p < 0.05). In experiment II animals were divided into two groups according to the eCG dose administered: 1000 and 1500 IU. Twenty hours before surgery females received a single i.v. dose of 8 microg of buserelin. Average number of follicles aspirated and COCs recovered was higher (p < 0.05) with the administration of 1500 IU of eCG. A larger number of expanded COCs were obtained from follicles > or =7 mm in diameter. We conclude that buserelin aids the recovery of a larger number of expanded COCs. Administration of 1500 IU of eCG produces a higher number of follicles for aspiration and number of COCs recovered.