Patterns of proliferation and cell differentiation during hepatic ontogeny in the alpaca.

The liver has multiple functions that change throughout ontogeny. South American camelids (SAC) have unique characteristics related to adaptation to extreme environments and metabolism. However, the process of hepatic cell differentiation has not been studied in any SAC. We study the patterns of cell differentiation and proliferation in the liver of the alpaca at different times of the ontogeny, excluding the hematopoietic components. Immunohistochemical techniques were performed in 66 specimens, including embryos, fetuses, neonates and adults. Supplementary analyses were performed by lectinhistochemistry. The hepatocytic differentiation was performed by the identification of Hepatocyte (Clone: ​​OCH1ES Dako®). It began in the specimens of 1.8-2.5 cm of crown to rump length (CRL), from Days 25-29 (ovulation = Day 0), continued during gestation and intensified towards its end. The cholangiocytic differentiation was performed by the identification of cytokeratin 7 (CK7, Dako®). It was manifested at the final of gestation (specimens of 28.4 cm CRL, from Day 223 onwards). Parenchymal cells underwent a process of gradual differentiation (differentiation of hepatocytes preceded that of cholangiocytes). Cell proliferation was observed along gestation using the nuclear proliferation antigen (PCNA) and Ki-67. Hepatic organogenesis in the alpacas shares similar differentiation and proliferation mechanisms with other altricial, but phylogenetically distant, species.

Mapping of the Early Intrauterine Morphogenesis in the Alpaca (Vicugna pacos): External Features and Development of the Cephalic Vesicle in Comparison with the Progressive Carnegie Scale.

In this study, we characterized the morphological aspects of the early development of the head of the alpaca (Vicugna pacos) and identified the main structures of the central nervous system during the first trimester of pregnancy. The topography and the cytoarchitecture of the fetal brain regions were described by histological analysis of the brain sections. We performed this analysis on alpaca embryos and fetuses presumably aged 20, 30, 45, and 90 days. For the description of the external body structures we considered the shape of the head, the development of the optic primordium, the dorsal curvature of the body, the limb buds, the umbilical cord and relative vessels, and the thickness and transparency of the skin. The prosencephalic, mesencephalic, and the rhomboencephalic vesicles were described by analyzing sagittal sections of the head. The present article provides the first progressive morphological and anatomical description of alpaca brain during early development. A detailed study represents an important basis to further understand the phases of prenatal development in this species, since information about alpaca embryology in incomplete and reproductive failure is a relevant factor. These data are important also for interspecies comparisons and application of reproductive biotechnologies. Anat Rec, 302:1226-1237, 2019. © 2018 Wiley Periodicals, Inc.

Morphology of alpaca (Vicugna pacos) embryos in the first third of pregnancy.

The breeding of South American camelids is the main economic activity of the high Andean region of South America and it, is potentially, the most profitable resource in of the Puna environmental conditions of the Puna. The duration of the gestation in alpaca is 339.7 ± 12 days. The objective of the present work was to macroscopically and microscopically describe the ontogenic development of the splanchnic cavities of the alpaca and to determine the gestational time in which the post-cranial ossification centers are observed in the embryos/fetuses of this species, from day 21 to 107 of gestation. The documentation of normal ontogenic development, which is vacant for this period, is of the utmost importance to understand the consequences of the alterations at the different gestational times, as well as for the estimation of the gestational age in the case of abortions. Forty-seven alpaca specimens of both sexes, at different times of their gestational development, collected during slaughter at local slaughterhouses of the Department of Huancavelica, Peru, were evaluated. Specimens were assigned to seven groups according to their morphological characteristics. The embryogenesis in the alpaca was characterized by a series of changes comparable to those occurring in other mammals with similar gestational periods. Despite these similarities, species differences were found in some organs as stomach, which are observed too in adult individuals.

Histological characterization of umbilical cord in alpaca (Vicugna pacos).

The histomorphometric features of umbilical cord constituents in seven foetuses of alpaca (Vicugna pacos) from Cerro de Pasco, Department, Peru, were determined. Sections of 2-5 cm of umbilical cord were fixed in 10% neutral buffered formalin and processed for light microscopy. Standard histological slides stained with haematoxylin and eosin, Masson's trichrome and Van Gieson's trichrome were obtained. Histologically, common features of umbilical artery and vein were observed as well as mucous connective tissue, some cell features that compound this tissue constituted by cells presented features of myofibroblasts. Among most important findings that were observed, the lumen of umbilical vein was obliterated into star-shaped form with the thinner umbilical artery wall; the smooth muscles and fibroblast were comparatively more in number in umbilical artery than that of umbilical vein, and the tunica media was larger in dimension than the tunica adventitia in umbilical vein. Conclusively, this histological study features an observation of the umbilical cord of alpaca foetuses and shows the similarity between them and those of other mammal species, including dromedaries and South American camelids.

Effect of oocyte maturation time, sperm selection method and oxygen tension on in vitro embryo development in alpacas.

We evaluated the effect of in vitro maturation time, sperm selection and oxygen tension on alpaca embryo development. In Experiment I, Cumulus Oocyte- Complexes (COCs) were obtained from abattoir ovaries and in vitro matured in TCM-199 for 24 (n = 217), 28 (215), or 32 h (223) at 38.5 °C, high humidity and 5% CO2 in air. Oocytes from 24 (n = 392), 28 (n = 456) or 32 (n = 368) h groups were in vitro fertilized with epididymal sperm and cultured in SOFaa at 38.5 °C, high humidity and 5% CO2, 5% O2 and 90% N2 for 7 days. Embryo development was evaluated on Day 2, 5 and Day 7 of in vitro culture (Day 0 = in vitro fertilization). In Experiment II, a 2 by 2-factorial design was used to determine the effect of sperm selection (Swim-up vs Percoll) and oxygen tension (20% vs 5%) during embryo culture and their interaction on embryo development. COCs were in vitro matured for 32 h at 38.5 °C and 5% CO2 in air and then in vitro inseminated with epididymal sperm processed by swim-up or Percoll. Zygotes were cultured in SOFaa + cumulus cells at 38.5 °C under 20 or 5% of O2 tension and high humidity for 7 days. A total of 235, 235, 253 and 240 oocytes were assigned to: swim-up+20 O2, swim-up+5 O2 or Percoll+20 O2, Percoll+5 O2, groups respectively. The proportion of oocytes reaching MII stage was highest after 32 h of in vitro maturation (P < 0.05). Blastocyst rate (29.1 ± 2.7%) was also highest for COCs matured for 32 h (Exp I). In Experiment II, Blastocysts rate (26.03 ± 4.7; 27.7 ± 4.3; 29.7 ± 3.8 and 27.6 ± 4.2% for swim-up+20 O2, swim-up+5 O2 or Percoll+20 O2, Percoll+5 O2, respectively) was not affected by sperm selection method (P = 0.8), oxygen tension (P = 0.9) or their interaction (P = 0.5).

Development of the Liver in Alpaca (Vicugna pacos): A Microscopic and Macroscopic Description.

South American camelids have several biological, morphological and behavioural adaptations that allow them to live in geographical areas dominated by high altitudes. The liver has hematopoietic functions during the prenatal life, which could be modified in response to the unfavorable habitat. However, there are no previous data on the prenatal development of the liver in these species. In the present work, a study on the macroscopic and microscopic morphology of the liver of the alpaca during ontogeny was performed. Forty-one animals ranging in age from 20 days of embryonic development to adults were studied. Macroscopic and microscopic observations were performed on samples subjected to different techniques. Less than 7-g specimens were studied with stereoscopic magnifying glass. The general characteristics of the prenatal liver are similar to those of other mammals, and the structures related to hematopoietic function follow an ontogenic pattern similar to that of previously studied precocial species. However, there are differences in morphology when compared to descriptions for the Old World camelids, including the absence of relation between the caudate lobe and the right kidney and the lack of interlobular connective tissue.

Double ovulation and occurrence of twinning in alpacas (Vicugna pacos).

Twin births are rare in alpacas despite the high incidence of double ovulation and are undesirable because they contribute to early and late pregnancy loss, abortion, and birth of nonviable neonates. The objective of the present study was to determine the incidence and outcome of twin pregnancy in double-ovulating alpacas by retrospective and prospective analysis. Data from double-ovulating females (N = 41) presented for pregnancy diagnosis were analyzed to determine pregnancy status at three stages after mating (14-16, 25-30, and 45-53 days). In a prospective study, adult reproductively sound alpacas (n = 21) were examined by ultrasonography to determine the incidence of multiple ovulations. A subset of those alpacas (n = 10) were euthanized either at 9 days (n = 5) or 14 days (n = 5) after a single mating and administration of GnRH to determine presence and number of embryos. A total of 31 cycles were included in the study to determine the incidence of multiple ovulations. In the retrospective study, twin pregnancies were identified between 25 and 30 days in 47.1% of double-ovulating females. There were more twins from bilateral ovulations (62.5%) than from unilateral ovulations (37.5%). Twin pregnancies were either reduced to a singleton (62.5%) or lost completely (37.5%). One set of twins remained viable until Day 52. In the prospective study, double ovulation occurred in 18.8% of the cycles. Two embryos were collected from two of the four double-ovulating females. In conclusion, twin conception is not rare in alpacas. A high rate of spontaneous reduction of twin pregnancies occurs before Day 45. However, a significant proportion of twin pregnancy may result in loss of both embryos. Determination of the number of ovulations (CL) at the time of early pregnancy diagnosis (14-16 days) should be an integral part of any pregnancy evaluation.

The entotympanic in late fetal Artiodactyla (Mammalia).

The entotympanic is a neomorphic component of the bulla tympanica of placental mammals. Ontogenetically, its rostral component seems to be derived from the tubal cartilage, whereas its caudal component is normally connected with the sheath of the tympanohyal; the present study indicates additional sources of the caudal entotympanic. The entotympanics develop in late fetal or early postnatal life as cartilaginous structures, but in most taxa they ossifiy endochondrally as "os bullae". This skeletal element is absent only in a few placental orders, among them the Artiodactyla. Because it is present in their sister taxa within the Scrotifera, it is likely to be reduced secondarily in the even-toed mammals. The study of histological serial sections of late fetal stages of several artiodactyl species shows that vestigial cartilaginous homologues of the entotympanics are invariably present, contrary to statements in the literature. In a few perinatal stages even secondary ossifications or calcifications of the entotympanic cartilages can be observed. The tubal cartilage of artiodactyls also continues into an anterior tegmen tympani (new term) that forms the floor of the fossa muscularis major.

Chronology of early embryonic development and embryo uterine migration in alpacas.

The objectives were to: (1) describe the chronology of early embryonic development from ovulation to entry into the uterus; and (2) to determine the timing of embryo migration to the left uterine horn when ovulation occurred from the right ovary. The experiment was conducted in Peru. Females (n = 132) were randomly assigned to 15 experimental groups. All females were mated to an intact male, given 50 µg GnRH im (Cystorelin) and ovulation time determined by transrectal ultrasonography, conducted every 6 hours, starting 24 hours postmating. Animals were slaughtered at a specific intervals postovulation and reproductive tracts were recovered and subjected to oviductal and uterine flushing for females slaughtered between 1 and 6 days postovulation (dpo; Day 0 = ovulation) and uterine flushing for females slaughtered from 7 to 15 dpo for recovery of oocytes/embryos. Season of mating did not influence the interval from mating to ovulation (winter: 29 ± 6 hours vs. summer: 30 ± 6 hours; P = 0.49). Ovulation rates for females mated during winter and summer were 92% versus 100%, respectively (P = 0.05). Fertilization rates for winter and summer mated females were 72% and 82% (P = 0.29). Unfertilized ova were not retained in the uterine tube. All embryos collected were in the uterine tube ipsilateral to the side of ovulation between 1 and 5 dpo. Embryos reached the uterus on 6 dpo. Embryos began to elongate on 9 dpo; at this time, 83% of embryos derived from right-ovary ovulations were collected from the left uterine horn. Embryos occupied the entire uterine cavity by 10 dpo. In conclusion, we characterized early embryo development and location of embryo during its early developmental stages in alpaca. This was apparently the first report regarding chronology of embryo development and migration to the left horn in alpaca which merits further investigation regarding its role in maternal recognition of pregnancy.

Factors influencing embryo transfer success in alpacas: a retrospective study.

Embryo transfer offers great advantages to South American camelid farmers to reach their breeding goals but the technology still plays a relatively minor role in comparison to other domestic farm animals like cattle. The aim of the present study was to analyse a data set of 5547 single or multiple ovulation embryo transfers performed in commercial alpaca farms in Australia to determine the factors that influence number and quality of embryos produced, embryo transfer success (percentage of crias born) and gestation length following transfer. Logistic binary regression identified the variables day of flushing after mating, embryo diameter, embryo quality, day of transfer after GnRH, and the age of the recipient to have significant impact on the outcome measure embryo transfer success. Transfer of smaller embryos or lower quality embryos resulted in decreased transfer success rates. Optimal days for obtaining embryos from donors were Days 8 and 9 after mating, optimal days for transfer into recipients were Days 7 and 8 after GnRH treatment. Age (>15 years) and body condition of recipients <2 also lowered transfer success rates, while the summer heat had no adverse impact. However, season did influence gestation length, while cria gender did not. In conclusion, results from the analysis of this very large dataset can underpin new recommendations to improve embryo transfer success in alpacas.

In vitro production of embryos in South American camelids.

Studies in reproductive biotechnology techniques have been minimal in South American camelids (SAC). Complex reproductive characteristics of these species contribute to slow progress. Nevertheless, some techniques, such as in vitro fertilization, intracytoplasmic sperm injection and nuclear transfer have been applied and have produced advances in knowledge on embryo environment and in vitro conditions necessary for development. Embryo production may have a high impact in both domestic and wild camelids population. Studies addressed to improve in vitro embryo production and oocyte collection could be a potential key to develop IVF and embryo production as a routine procedure in camelids.

Embryo transfer in domestic South American camelids.

Intraspecific and interspecific embryo transfer in domestic South American camelids is developing into a well-established technique. Reports reveal many benefits of using reproductive biotechnologies to allow rapid propagation of alpacas and llamas of high genetic merit (e.g., high fiber quality, preserve color variation). The objective of this review is to provide up-to-date information about embryo transfer in domestic South American camelids. Specific information is provided on criteria for male selection, donor and recipient synchronization, the practice of single- vs. super-ovulation protocols, embryo recovery and transfer techniques, advances in cryopreservation of embryos, results of intra- and inter-specific transfer, and the future of the embryo transfer in domestic South American camelids.

In vitro embryo production in llamas (Lama glama) from in vivo matured oocytes with raw semen processed with Androcoll-E using defined embryo culture media.

The aim of this study was to carry out in vitro fertilization using spermatozoa selected with Androcoll-E™ and to evaluate the efficiency of the culture medium DMEM-F12 for in vitro embryo development in the llama. Twelve adult females from 18 superstimulated (67%) were used as oocyte donors. They were superstimulated with 1500 IU of eCG and after 5 days, received a single dose of buserelin. Twenty hours post-injection, follicular aspiration was conducted by flank laparotomy. Semen collections were performed under general anesthesia by electroejaculation of the male. The ejaculates were processed with a solution of collagenase (0.1%) and an Androcoll-E™ column was used to improve the sample. Sixty nine COCs were recovered from 79 aspirated follicles (87% recovery). Only expanded COCs were used (n = 67); they were randomly placed in groups of 1-5 in Fertil-TALP and the sperm suspension (20 × 10(6) live spermatozoa/ml) was added to each fertilization microdroplet. After 24 h, they were randomly placed in one of two culture media: SOF (n = 34) or DMEM-F12 (n = 33) and incubated for 6 days in humidified atmosphere of 5% CO(2) , 5% O(2) and 90% N(2) at 38°C. The blastocyst rate was 20% (7/34) in SOF medium (3 hatched, 2 expanded and 2 early blastocysts) and 15% (5/33) in DMEM medium (all expanded blastocysts). In conclusion, using Androcoll-E™ it is possible to select good quality spermatozoa from llama ejaculates for in vitro fertilization and to produce blastocysts in DMEM-F12 medium. This is also the first time that hatched llama blastocysts have been produced after culture in a defined medium such as SOFaa.

Esophageal anatomy of the llama (Lama glama) / Anatomía del esófago de la llama (Lama glama)

This study describes gross, microscopic and muscle fiber anatomy of the esophagus of the llama, Lama glama. The esophagus was studied grossly in twenty-five adult llamas and a subset of ten with normal esophageal physiology was used for the microanatomic studies. Esophageal length was 122 +/- 7 cm with two-thirds of the length in the neck and the remainder in the thorax, consistent with the long neck of the llama. Esophageal diameter increased steadily from 2.5 +/- 0.3 cm in the cranial cervical region to 3.9 +/- 0.8 cm in the caudal thoracic region. The mucosal epithelium was keratinized stratified squamous and there were abundant submucosal glands throughout the esophagus. The entire muscularis of the esophagus was striated muscle in two general layers but also with a somewhat random orientation of fibers. The tunica muscularis steadily increased in thickness from 3.43 +/- 0.30 mm in the cranial cervical region to 4.39 +/- 0.39 mm in the middle thoracic region. In the llama Type 2 muscle fibers predominated in the esophageal musculature, with the percentage of Type 1 fibers increasing from 1 percent cranially to 33 percent in the caudal thoracic region of the esophagus. This study of the normal llama esophagus enhances our knowledge of this species and provides the basis for future study of pathological conditions of the esophagus.

Este estudio describe la anatomía morfológica, microscópica, y tipo de fibra muscular del esófago de la llama, Lama glama. Estudiamos la anatomía morfológica del esófago, con fisiología normal, en 25 llamas adultas y, adicionalmente, en 10 de ellas la anatomía microscópica. La longitud del esófago fue 122 +/- 7 cm con dos tercios en el cuello y un tercio en el tórax. El diámetro del esófago aumentó de 2,5 +/- 0,3 cm en la región craneal del cuello y a 3,9 +/- 0,8 cm en la región caudal del tórax. El epitelio de la mucosa eera escamoso estratificado queratinizado y la submucosa contenía abundantes glándulas a lo largo de todo el esófago. La muscular entera del esófago se compuso de músculo esquelético en más o menos dos capas, pero con algunas fibras orientadas al azar. La muscular aumentó de 3,43 +/- 0,30 mm en la región craneal del cuello a 4,39 +/- 0,39 mm en la región media del tórax. Fibras musculares Tipo 2 predominaron en la muscular. El porcentaje de fibras Tipo 1 aumentó de 1 por ciento al inicio del esófago a 33 por ciento en la región caudal torácica. Este estudio del esófago normal de la llama ofrece más información sobre la anatomía de la llama y proporciona una base para futuros estudios de patologías esofágicas.

Ovarian response and embryo production in llamas treated with equine chorionic gonadotropin alone or with a progestin-releasing vaginal sponge at the time of follicular wave emergence.

The objective of the study was to compare the ovulatory response and embryo production in llamas (Lama glama) treated with a single dose of equine chorionic gonadotropin (eCG) alone or combined with intravaginal medroxyprogesterone acetate (MPA) at the time of follicular wave emergence. Llamas with a growing follicle >or=7 mm in diameter were assigned to one of the following groups: (1) Control (n=28): Nonstimulated llamas were mated and embryos were collected 7 d after mating. (2) eCG (n=32): Llamas were given 5mg luteinizing hormone (LH) (Day 0) to induce ovulation, 1000 IU eCG on Day 2, a luteolytic dose of prostaglandin F(2alpha) on Day 6, mating on Day 7, and embryo collection on Day 14. (3) eCG+MPA (n=34): Llamas were treated as those in the eCG group, but a sponge containing 60 mg MPA was placed intravaginally from Days 2 to 6. Llamas that did not respond to synchronization or superstimulation were excluded, leaving data from n=26, 26, and 27 in the control, eCG, and eCG+MPA groups, respectively, for statistical analysis. The mean (+/-SD) number of follicles>7 mm at the time of mating was greatest in the eCG group, intermediate in the eCG+MPA group, and lowest in the control group (16.6+/-5.3, 12.9+/-3.7, and 1.0+/-0.0, respectively, P<0.001). The number of corpora lutea was similar between eCG and eCG+MPA groups (10.1+/-2.9 and 8.6+/-3.7, respectively); both were higher (P<0.001) than in controls (0.9+/-0.3). The number of embryos did not differ significantly between the eCG and eCG+MPA groups (4.8+/-2.8 and 3.5+/-3.0, respectively), but both were higher (P<0.001) than in the controls (0.7+/-0.4). In conclusion, eCG, with or without MPA effectively induced a superovulatory response and multiple embryo production in llamas.

Músculo romboides supraescapular de la Llama (Lama glamá) / Rhomboideus suprascapularis muscle of the llama (Lama glama)

El músculo romboides (m. rhomboideus) forma parte de la sinsarcosis que une la cintura del miembro torácico con el esqueleto axil en los mamíferos domésticos. En estas especies, está integrado por los músculos romboides cervical (m. rhomboideus cervicis) y romboides torácico (m. rhomboideus thoracis), siendo imposible establecer el límite entre ellos, a diferencia de lo que sucede en el Hombre. Los carnívoros en general, el cerdo y el conejo presentan, además de las partes mencionadas, el músculo romboides de la cabeza (m. rhomboideus capitis). En la llama, la porción cefálica está ausente y la cervical pobremente desarrollada. Los autores proponen sumar a las porciones cervical y torácica del músculo romboides de este camélido sudamericano, al músculo romboides supraescapular (m. rhomboideus suprsacapularis), descrito por Lesbre (1903), en el camello y el dromedario. En este trabajo se establecen las inserciones, dimensiones e inervación del músculo romboides supraescapular de la llama, elementos que permiten definirlo como otra porción del complejo muscular romboideo en dicha especie. Además, se postula su acción como elevador de la escápula, dirigiéndola craneal y dorsalmente.

The rhomboideus muscle of the domestic mammals is part of the muscular set that joins the scapular waist to the axial skeleton. In these animals, the forenamed muscle has a cervical portion (m. rhomboideus cervicicis), and a thoracic portion (m. rhomboideus thoracis). Unlike in the man, these parts cannot be separated. In addition, carnivores, pigs and rabbits also have a cephalic portion (m. rhomboideus capitis). In the llamas, the cephalic portion is absent, and the cervical part is poorly developed. The authors propose to add a suprascapular portion (m. rhomboideus suparsacapularis), first described by Lesbre, 1903 in camels, to the Rhomboideus muscular complex of the llama. In this study, the authors describe the length, insertions, and inervation of the Rhomboideus Suprascapular muscle of the llama in order to define it as another portion of the Rhomboideus muscular complex. Moreover, the forenamed muscle is proposed as a scapular elevator.

Conformation and anatomical relations of the liver of llama (Lama glama).

Morphological studies of the liver of the llama are structural supportive to the clinical practice, surgery and specific diagnostic techniques. The aims of this study were first to determine the location of the organ and the direction of its major axis to project it to the abdominal wall, identifying visible and palpable bony references. Secondly, to characterize and determine anatomical relations of the surfaces, borders and angles of the llama liver, as well as, of its lobulation. Twenty adult llamas of both sexes and two foetuses of 6.5- and 7-month-old were used. Llama liver is a post-diaphragmatic organ located in the cranial abdominal region, in the right hypochondrium, in relationship with the last six ribs. Dorsally, it can exceeds the last (twelfth) rib. Its major axis presents a cranio-ventral bent. Its shape is irregularly triangular. It presents two surfaces (parietal and visceral), three borders (cranial, caudal and ventral) and three angles (dorsal, cranial and caudal).

In vitro production of llama (Lama glama) embryos by intracytoplasmic sperm injection: effect of chemical activation treatments and culture conditions.

Assisted reproductive technologies in the llama (Lama glama) are needed to provide alternative methods for the propagation, selection and genetic improvement; however, recovery of adequate quantity and quality of spermatozoa for conventional IVF is problematic. Therefore, an effort was made to adapt the intracytoplasmic sperm injection (ICSI) procedure for the in vitro production of llama embryos. The specific objectives of this study were: (1) to determine in vitro maturation rates of oocytes recovered by transvaginal ultrasound-guided oocyte aspiration (TUGA) or flank laparotomy; (2) to evaluate the effects of activation treatments following ICSI; (3) to evaluate the development of llama ICSI embryos in CR1aa medium or in an oviduct cell co-culture system. Llamas were superstimulated by double dominant follicle reduction followed by oFSH administered in daily descending doses over a 3-day interval. Oocytes were harvested by flank laparotomy or TUGA and matured in vitro for 30 h. Mature oocytes were subjected to ICSI followed by no chemical activation (Treatment A), ionomycin only (Treatment B) or ionomycin/DMAP activation (Treatment C). More oocytes were recovered by flank laparotomy procedure compared with TUGA (94% versus 61%, P<0.05) and a greater number of oocytes harvested by flank laparotomy reached the metaphase-II stage (77% versus 44%, P<0.05). After ICSI, the proportion of cleaved and 4-8-cell stages embryos was significantly greater when injected oocytes were activated with ionomycin/DMAP combination (63% and 38%, respectively, P<0.05). The co-culture of ICSI embryos with llama oviduct epithelial cells resulted in progression to morula (25%) and blastocyst (12%) stages; whereas, all embryos cultured in CR1aa medium arrested at the 8-16-cell developmental stage.

Fetal brain hypometabolism during prolonged hypoxaemia in the llama.

In this study we looked for additional evidence to support the hypothesis that fetal llama reacts to hypoxaemia with adaptive brain hypometabolism. We determined fetal llama brain temperature, Na(+) and K(+) channel density and Na(+)-K(+)-ATPase activity. Additionally, we looked to see whether there were signs of cell death in the brain cortex of llama fetuses submitted to prolonged hypoxaemia. Ten fetal llamas were instrumented under general anaesthesia to measure pH, arterial blood gases, mean arterial pressure, heart rate, and brain and core temperatures. Measurements were made 1 h before and every hour during 24 h of hypoxaemia (n = 5), which was imposed by reducing maternal inspired oxygen fraction to reach a fetal arterial partial pressure of oxygen (P(a,O(2))) of about 12 mmHg. A normoxaemic group was the control (n = 5). After 24 h of hypoxaemia, we determined brain cortex Na(+)-K(+)-ATPase activity, ouabain binding, and the expression of NaV1.1, NaV1.2, NaV1.3, NaV1.6, TREK1, TRAAK and K(ATP) channels. The lack of brain cortex damage was assessed as poly ADP-ribose polymerase (PARP) proteolysis. We found a mean decrease of 0.56 degrees C in brain cortex temperature during prolonged hypoxaemia, which was accompanied by a 51% decrease in brain cortex Na(+)-K(+)-ATPase activity, and by a 44% decrease in protein content of NaV1.1, a voltage-gated Na(+) channel. These changes occurred in absence of changes in PARP protein degradation, suggesting that the cell death of the brain was not enhanced in the fetal llama during hypoxaemia. Taken together, these results provide further evidence to support the hypothesis that the fetal llama responds to prolonged hypoxaemia with adaptive brain hypometabolism, partly mediated by decreases in Na(+)-K(+)-ATPase activity and expression of NaV channels.

Vasodilator tone in the llama fetus: the role of nitric oxide during normoxemia and hypoxemia.

The fetal llama responds to hypoxemia, with a marked peripheral vasoconstriction but, unlike the sheep, with little or no increase in cerebral blood flow. We tested the hypothesis that the role of nitric oxide (NO) may be increased during hypoxemia in this species, to counterbalance a strong vasoconstrictor effect. Ten fetal llamas were operated under general anesthesia. Mean arterial pressure (MAP), heart rate, cardiac output, total vascular resistance, blood flows, and vascular resistances in cerebral, carotid and femoral vascular beds were determined. Two groups were studied, one with nitric oxide synthase (NOS) blocker N(G)-nitro-L-arginine methyl ester (L-NAME), and the other with 0.9% NaCl (control group), during normoxemia, hypoxemia, and recovery. During normoxemia, L-NAME produced an increase in fetal MAP and a rapid bradycardia. Cerebral, carotid, and femoral vascular resistance increased and blood flow decreased to carotid and femoral beds, while cerebral blood flow did not change significantly. However, during hypoxemia cerebral and carotid vascular resistance fell by 44% from its value in normoxemia after L-NAME, although femoral vascular resistance progressively increased and remained high during recovery. We conclude that in the llama fetus: 1) NO has an important role in maintaining a vasodilator tone during both normoxemia and hypoxemia in cerebral and femoral vascular beds and 2) during hypoxemia, NOS blockade unmasked the action of other vasodilator agents that contribute, with nitric oxide, to preserving blood flow and oxygen delivery to the tissues.