Seeking temporal refugia to heat stress: increasing nocturnal activity despite predation risk.

Flexibility in activity timing may enable organisms to quickly adapt to environmental changes. Under global warming, diurnally adapted endotherms may achieve a better energy balance by shifting their activity towards cooler nocturnal hours. However, this shift may expose animals to new or increased environmental challenges (e.g. increased predation risk, reduced foraging efficiency). We analysed a large dataset of activity data from 47 ibex (Capra ibex) in two protected areas, characterized by varying levels of predation risk (presence versus absence of the wolf-Canis lupus). We found that ibex increased nocturnal activity following warmer days and during brighter nights. Despite the considerable sexual dimorphism typical of this species and the consequent different predation-risk perception, males and females demonstrated consistent responses to heat in both predator-present and predator-absent areas. This supports the hypothesis that shifting activity towards nighttime may be a common strategy adopted by diurnal endotherms in response to global warming. As nowadays different pressures are pushing mammals towards nocturnality, our findings emphasize the urgent need to integrate knowledge of temporal behavioural modifications into management and conservation planning.

Whole-genome SNP genotyping unveils ancestral and recent introgression in wild and domestic goats.

After the domestication of goats around 10,000 years before the present (BP), humans transported goats far beyond the range of their wild ancestor, the bezoar goat. This brought domestic goats into contact with many wild goat species such as ibex and markhor, enabling introgression between domestic and wild goats. To investigate this, while shedding light on the taxonomic status of wild and domestic goats, we analysed genome-wide SNP data of 613 specimens from 14 taxonomic units, including Capra hircus, C. pyrenaica, C. ibex (from Switzerland, Austria, Germany and Slovenia), C. aegagrus aegagrus, C. a. cretica, C. h. dorcas, C. caucasica caucasica, C. c. severtzovi, C. c. cylindricornis, C. falconeri, C. sibirica sibirica, C. s. alaiana and C. nubiana, as well as Oreamnos americanus (mountain goat) as an outgroup. To trace gene flow between domestic and wild goats, we integrated genotype data of local goat breeds from the Alps as well as from countries such as Spain, Greece, Türkiye, Egypt, Sudan, Iran, Russia (Caucasus and Altai) and Pakistan. Our phylogenetic analyses displayed a clear separation between bezoar-type and ibex-type clades with wild goats from the Greek islands of Crete and Youra clustered within domestic goats, confirming their feral origin. Our analyses also revealed gene flow between the lineages of Caucasian tur and domestic goats that most likely occurred before or during early domestication. Within the clade of domestic goats, analyses inferred gene flow between African and Iberian goats. The detected events of introgression were consistent with previous reports and offered interesting insights into the historical relationships among domestic and wild goats.

Facilitated introgression from domestic goat into Alpine ibex at immune loci.

Hybridization can result in the transfer of adaptive genetic material from one species to another, known as adaptive introgression. Bottlenecked (and hence genetically depleted) species are expected to be particularly receptive to adaptive introgression, since introgression can introduce new or previously lost adaptive genetic variation. The Alpine ibex (Capra ibex), which recently recovered from near extinction, is known to hybridize with the domestic goat (Capra aegagrus hircus), and signals of introgression previously found at the major histocompatibility complex were suggested to potentially be adaptive. Here, we combine two ancient whole genomes of Alpine ibex with 29 modern Alpine ibex genomes and 31 genomes representing six related Capra species to investigate the genome-wide patterns of introgression and confirm the potential relevance of immune loci. We identified low rates of admixture in modern Alpine ibex through various F statistics and screening for putative introgressed tracts. Further results based on demographic modelling were consistent with introgression to have occurred during the last 300 years, coinciding with the known species bottleneck, and that in each generation, 1-2 out of 100 Alpine ibex had a domestic goat parent. The putatively introgressed haplotypes were enriched at immune-related genes, where the adaptive value of alternative alleles may give individuals with otherwise depleted genetic diversity a selective advantage. While interbreeding with domestic species is a prevalent issue in species conservation, in this specific case, it resulted in putative adaptive introgression. Our findings highlight the complex interplay between hybridization, adaptive evolution, and the potential risks and benefits associated with anthropogenic influences on wild species.

Human social buffer in goats and dogs.

The primary goal of this study was to explore the social buffering effect that humans offer to goats and dogs with limited exposure to human socialization, particularly in situations involving interactions with unfamiliar humans. A total of 13 dogs and 14 goats were selected for the study, all of which had limited prior socialization with humans. Each animal was placed in a testing room with unfamiliar humans for 15 min. Three experimenters aimed to establish a comfortable environment, encouraging social interaction by offering food to the animals and assessing the animals' willingness to accept food and their response to being approached and petted. If both conditions were satisfied, the animals were classified as "social". If one or none of the conditions were met, the animals were classified as "not social". Cortisol levels were measured by collecting blood samples before and after the test. Non-parametric tests together with a GzLM showed that the effect of human social buffering in goats was different in comparison to dogs: goats exhibited higher cortisol levels after the test, while dogs did not show a significant change. Further analysis demonstrated that "social" goats had a lower likelihood of experiencing significant changes in cortisol levels than dogs. Thus, once human interactions are accepted, both species could benefit from social buffering. In summary, this study enhances our understanding of how dogs and goats respond to social interactions with humans in the social buffering effect.

Morpometric and molecular characterization of Surguli goat through CO1 gene in district Kohat.

The present study was designed with the aim to study morphometric characterization as well as phylogeny and diversity of the local Surguli goat at their breeding tract district Kohat through mitochondrial DNA region, i.e., Cytochrome C Oxidase Subunit One (CO1) gene. Morphometric data and blood samples were collected from thirty (30) pure goats. Morphometric analysis showed that sex had significant effect (p < 0.05) on body weight, body length, hearth girth and horn length while no significant effect (p > 0.05) was observed for other characteristics. The results also indicated that age had significant effect (p < 0.05) on height at rump, ear length, horn length and tail length while no significant effect (p > 0.05) was observed for other characteristics. The phylogenetic analysis through CO1 nucleotide sequences within nucleotide range 1-767 showed nine polymorphic sites segregating into eight haplotypes. The mean intraspecific diversity and mean interspecific diversity were calculated as 0.23 and 2.36%, respectively. Phylogenetic tree revealed that Capra Ibex and native Surguli goat have common ancestors. The morphometric and molecular results obtained from the present study can be exploited as a selection tool for breeding and overall improvement.

Pharmacokinetic profile and physiological effects of oral and compounded intravenous gabapentin in goats.

OBJECTIVE: To determine the pharmacokinetics and physiological effects following oral and intravenous (IV) administration of gabapentin in goats. STUDY DESIGN: Prospective, crossover study with a 3 week washout period between treatments. ANIMALS: A total of eight healthy, client-owned, female goats. METHODS: Gabapentin (10 mg kg-1) was administered to goats either orally or IV. Gabapentin concentrations were measured in serum samples collected 0-96 hours post-administration using liquid chromatography-quadrupole time-of-flight mass spectrometry. Heart rate, respiratory rate, blood pressure and temperature were recorded before and throughout the study. Correlations of the mean serum concentrations of gabapentin to those of each physiological parameter were determined using the Pearson method. RESULTS: The mean and standard deviation of oral bioavailability for gabapentin was 60.9 ± 11.2%. Maximum serum concentration of gabapentin was lower following oral (1.19 ± 0.29 µg mL-1) than after IV administration (59.76 ± 14.38 µg mL-1, p < 0.0001). Half-lives were longer following PO (8.18 ± 0.57 hours) than after IV administration (1.79 ± 0.06 hours, p < 0.0001). Time to maximum concentration was 6.86 ± 2.27 hours following oral administration. Heart rate was inversely correlated with serum gabapentin concentrations. Slight ataxia was observed in three animals, and one became recumbent following IV gabapentin. CONCLUSIONS AND CLINICAL RELEVANCE: Gabapentin is well-absorbed following oral administration to goats but yielded significantly lower serum concentrations than the IV route. The longer half-life of gabapentin following oral than after IV administration may result from prolonged absorption throughout the caprine gastrointestinal tract. IV gabapentin may cause slight ataxia in some goats.

Participatory investigation of goat farmers' breeding practices, trait preference, and selection criteria in Burkina Faso.

The community-based breeding program (CBBP) is an innovative approach recommended for genetic improvement and sustainable use of animal genetic resources in extensive farming systems. Successful implementation of this approach requires an understanding of the characteristics of production systems, breeding objectives, and farmers' trait preference. This study aimed to identify the selection criteria of goat farmers in rural areas of Burkina Faso and their potential implications in establishing CBBP. Following focus group discussions, a well-structured questionnaire was designed and administered to 372 randomly selected goat farmers in two different agro-ecological zones. A list of traits obtained during focus group discussions was provided to farmers individually, and they were asked to rank the ones they preferentially use to select breeding animals. Statistical tests were conducted to compare data between the two agro-ecological zones. The results showed that the average goat flock per household was higher (P < 0.05) in the Sudanian (15.68 ± 13.76), compared to the Sudano-Sahelian area (12.93 ± 13.3). Adult females were the dominant age-sex group in both areas. Reasons for culling, keeping breeding bucks, and castration practice were significantly different (P < 0.05) among agro-ecological zones. The most important common criterion for selection in the two zones was body size, coat color, and growth rate for the bucks and does, while fertility (0.06) parameters including twining ability (0.18), kidding frequency (0.11), and mothering ability (0.15) were furthermore considered for breeding does selection. These findings provide valuable insights for developing CBBPs tailored to goat production in the study areas.

Genomic Characteristics of Emerging Intraerythrocytic Anaplasma capra and High Prevalence in Goats, China.

Anaplasma capra is an emerging tickborne human pathogen initially recognized in China in 2015; it has been reported in ticks and in a wide range of domestic and wild animals worldwide. We describe whole-genome sequences of 2 A. capra strains from metagenomic sequencing of purified erythrocytes from infected goats in China. The genome of A. capra was the smallest among members of the genus Anaplasma. The genomes of the 2 A. capra strains contained comparable G+C content and numbers of pseudogenes with intraerythrocytic Anaplasma species. The 2 A. capra strains had 54 unique genes. The prevalence of A. capra was high among goats in the 2 endemic areas. Phylogenetic analyses revealed that the A. capra strains detected in this study were basically classified into 2 subclusters with those previously detected in Asia. Our findings clarify details of the genomic characteristics of A. capra and shed light on its genetic diversity.

Laparoscopic left hepatectomy in a goat as a training model for laparoscopic anatomic liver resection: results of training courses with a total of 70 goats.

BACKGROUND: To create a suitable animal model for the training of laparoscopic anatomic liver resection, we performed left hepatectomy using a goat and found its suitability. We have since started using goats for wet-lab training and have gradually standardized the relevant procedures. Herein, we report our standardized training procedures using a goat and discuss its feasibility as a novel training model. METHODS: The standardized wet-lab training courses of laparoscopic liver resection conducted on 62 tables with a total of 70 goats were reviewed. The training course began by encircling the hepatoduodenal ligament for the Pringle maneuver, which was repeated during the parenchymal dissection. Following partial liver resection of the left lateral section, left hepatectomy was performed by a standardized procedure for humans in which the liver was split, exposing the entire length of the middle hepatic vein trunk from the dorsal side after extrahepatic transection of the left Glissonean pedicle. If a goat deceased before initiating left hepatectomy, the training was restarted with a new goat. The surgical procedures were performed by surgeons of varying skill levels. RESULTS: A total of 184 surgeons including 10 surgical residents participated in the training. Partial liver resection was initiated in 62 tables, with 8 (13%) dying during or after the procedure of partial liver resection. Subsequently, left hepatectomy was initiated in 61 and completed in 59 tables (98%), regardless of whether the goat survived or deceased, and was not completed in 2 tables (3%) due to time limitation. In 14 tables (23%), the goats deceased during the procedure, however, the procedure was completed. The causes of death were multifactorial, including massive bleeding, reperfusion injury after the Pringle maneuver, and carbon dioxide gas embolism. CONCLUSIONS: Left hepatectomy in a goat is useful as a training model for laparoscopic anatomic liver resection.

Maternal genetic diversity of ancient goats in Finland and Estonia and comparison with extant northern European goat breeds.

Countries in the northern Baltic Sea region have been historically linked for thousands of years, and animal husbandry is one of the many information sources that enables the study of historical connections. Although goat husbandry in this part of Europe has been evidenced by scant archaeological materials, zooarchaeological and historical evidence has revealed its continuity, at least since the Late Iron Age. To explore the historical relationship between goat lineages and investigate affinities between the past and present-day populations in the Baltic Sea region, we analysed a 476-bp fragment of the mitochondrial DNA control region in 14 ancient goats from Finland and Estonia and 10 extant goats from Finland. The results revealed high mitochondrial diversity among the ancient goats. Two maternal lineages were shared between the Late Iron Age and medieval individuals from Finland and Estonia. Moreover, ancient Finnish and Estonian goats showed maternal affinity to extant Finngoat and Swedish Landrace breeds. Overall, the analysis of maternal goat lineages confirmed tight historical connections in the region.

Association between the copy number variation of CCSER1 gene and growth traits in Chinese Capra hircus (goat) populations.

Recently, Coiled-coil serine-rich protein 1 (CCSER1) gene is reported to be related to economic traits in livestock, and become a hotspot. In our study, we detected CCSER1 gene CNV in 693 goats from six breeds (GZB, GZW, AN, BH, HG, TH) by quantitative real-time PCR (qPCR) and the association analysis between the types of CNV and growth traits. Then, CCSER1 gene expression pattern was discovered in seven tissues from NB goats. Our results showed that the CCSER1 gene copy numbers were distributed differently in the aforementioned six breeds. The type of CCSER1 gene CNV was significantly associated with body weight and heart girth traits in GZW goat, in which individuals with deletion type were dominant in body weight trait (P < 0.05), while the normal type individuals were more advantageous in heart girth trait (P < 0.01); and there was a significant association with heart girth in TH goat (P < 0.05), which normal type was the dominant one. The expression profile revealed that CCSER1 gene has the highest level in the lung, followed by the small intestine and heart. In conclusion, our result is dedicated to an in-depth study of the novel CCSER1 gene CNV site and to provide essential information for Chinese goats molecular selective breeding in the future.

A retinoscopic survey of donkeys and goats.

PURPOSE: Assess the refractive states of donkeys and goats. METHODS: Forty-two donkeys and 28 goats were enrolled. The mean ± SD ages were 7.68 ± 7.33 years for donkeys and 4.26 ± 2.33 years for goats. Seven donkeys and one goat were <6 months old. Retinoscopy was performed in alert animals, following cycloplegia in goats but not in donkeys. Normality was determined using the Kolmogorov-Smirnov test. The two primary meridians and two eyes were compared using Pearson's correlation and paired Student's t-tests. The association between refractive states and age was examined using one-way ANOVA in donkeys and a paired Student's t-test in goats. One-sample t-tests were conducted to assess if the refractive error distributions were significantly different from "0". RESULTS: The mean ± SD spherical equivalent (SE) refractive errors of the right and left donkey eyes were -0.80 ± 1.03 D and -0.35 ± 0.95 D, respectively. The majority (86%) of the donkeys had an astigmatic refraction and eight (19%) had anisometropia. The mean SE refractive errors of the right and left goat eyes were -0.15 ± 1.1 D and -0.18 ± 1.2 D, respectively. The majority (54%) of the goat eyes had an astigmatic refraction and five (18%) had anisometropia. The right and left eye SE refractive errors were positively correlated in both species (both p = .9). Age was not correlated with refractive error in both donkeys (p = .09) and goats (p = .6). CONCLUSIONS: Both goats and donkeys are emmetropic.

Validation of double freezing protocol for Beetal buck (Capra hircus) spermatozoa.

The study aimed to validate the double versus single freezing protocol for Beetal buck (Capra hircus) spermatozoa in tris-citric acid (TCA) based extender both in terms of quality and fertilization potential. Computer-assisted sperm motion and kinematic (CASA) variables, i.e. total (%), and progressive motilities (TM and PM, %) and rapid velocity (RV, %), average path (VAP, µm/s), straight line (VSL, µm/s) and curved line velocities (VCL, µm/s), straightness, (VSL/VAP, %) and linearity, (VSL/VCL, %) as well as supra-vital plasma membrane integrity (SV-PMI, %), mitochondrial membrane potential (MMP, %), viable/intact acrosome (V-IACR, %) and DNA integrity (DNA-I, %) had significantly greater values (p < .05) during single freeze-thawing as compared with the double freeze-thawing at 0, 30, 90, 150 and 210 days, respectively. All CASA and other assays alone did not show significant differences (p > .05) between both freeze-thaw cycles at all treatment durations, respectively. No statistical significance (p > .05) was observed for the in vivo fertility between single (n = 84/141 = 59.72%) and double freeze-thawing (n = 72/136 = 52.9%) cycles, respectively. In conclusion, sperm motion, kinematics, plasma membrane, acrosome, mitochondria and DNA integrities and in vivo fertility are acceptable after the double freezing protocol despite being lower than after one freeze cycle in Beetal buck.

Whole-genome sequencing to identify candidate genes for litter size and to uncover the variant function in goats (Capra hircus).

To select candidate genes for goat prolificacy, we managed six multi- and six single-kid female goats at the same feeding level and in the same management mode over a 4-year period. These goats showed stable differences in litter size over five continuous parturition records. Whole-genome re-sequencing was used in all 12 to select candidate genes, namely, AURKA, ENDOG, SOX2, RORA, GJA10, RXFP2, CDC25C, and NANOS3, by the strength of their differentiation signals. Most of the selected genes were enriched in the coiled coil process and ovarian development, which suggests that the coiled coil process has a potential regulatory effect on fecundity. Detection of the distribution of variants and association analyses with litter size in 400 goats showed that NANOS3 exon mutations may lead to a transformation of the protein structure. The variation in CDC25C, ENDOG, and NANOS3 showed a significant association with litter size. These results can contribute to the improvement of reproduction traits in the artificial breeding of goats.

Reticular contraction frequency and ruminal gas dome development in goats do not differ between grass and browse diets.

In investigations of differences between ruminant species feeding on browse or grass, it is often unclear whether observed differences are animal- or forage-specific. Ruminant species have been classified as 'moose-type', with little rumen content stratification, or 'cattle-type' with a distinct rumen contents stratification, including a gas layer. To which extent putative differences in forestomach motility are involved in these patterns is unknown. Using sonography, we investigated the frequency of reticular contractions and the stratification of rumen contents in goats fed exclusively on grass hay (n = 6) or dried browse (n = 5) directly after feeding, and after another 6 and 12 h with no access to feed. The frequency of reticular contractions decreased from immediately after feeding (1.8 ± 0.3 min-1 ) to 6 h afterwards (1.2 ± 0.2 min-1 ) and then remained constant, with no difference between diets. A gas dome became more visible over time, but neither its incidence nor its extent differed between diets. The results are in accord with classifying goats as 'cattle-type' in terms of their digestive physiology, and they add to a growing body of evidence that differences in digestive physiology between ruminant species are more due to species characteristics than different kinds of ingested forages.

The first molecular detection of Anaplasma capra in domestic ruminants in the central part of Turkey, with genetic diversity and genotyping of Anaplasma capra.

Tick-borne diseases have been an increasing threat to human and animal health all over the world. Anaplasmosis is one of the emerging tick-borne diseases and has zoonotic potential. A new novel species, which was detected in China in 2010-2012 and provisionally named Anaplasma capra in 2015, causes zoonotic infections and infects many different animal species. In this study, we investigated the presence of A. capra in domestic ruminants from Turkey. A total of 468 blood samples (cattle, sheep, and goat) were examined by the gltA gene-specific nested polymerase chain reaction, revealing the presence of A. capra in six samples (1.28%): one of them from cattle (0.41%) and the other five from sheep (3.22%). According to DNA sequences results of the gltA gene, A. capra isolates identified in the present study were shown high nucleotide similarity with A. capra isolates detected from different hosts. However, the nucleotide differences were detected in the same nucleotide positions between A. capra isolates. For this reason, we thought that at least two different A. capra genotypes could be circulating in the world. As a result, it is seen that A. capra, which was determined to be a new species with zoonotic potential, was revealed in European and Asian countries and in different hosts. In order to raise awareness about human anaplasmosis infections, it is important to reveal the prevalence of the species in the world. The emergence of A. capra in Turkey reveals the need for a re-evaluation of the human and animal health risk analysis in terms of anaplasmosis.

Behavioural heat-stress compensation in a cold-adapted ungulate: Forage-mediated responses to warming Alpine summers.

Alpine large herbivores have developed physiological and behavioural mechanisms to cope with fluctuations in climate and resource availability that may become maladaptive under climate warming. We tested this hypothesis in female Alpine ibex (Capra ibex) by modelling annual and daily movement and activity patterns in relation to temperature, vegetation productivity and reproductive status based on bio-logging data and climate change projections. In summer, ibex moved upslope, tracking the green wave. Ibex decreased diel activity sharply above a threshold temperature of 13-14°C, indicating thermal stress, but compensated behaviourally by foraging both earlier and later in the day, and by moving further upslope than on cooler days, especially reproductive females. This critical temperature will be exceeded three times as often under climate change projections. Under such scenarios, the altitudinal extent of the area will limit the available habitat providing thermal shelter, potentially impacting performance and population distribution of this emblematic mountain ungulate.

Transcriptome sequencing and multi-plex imaging of prostate cancer microenvironment reveals a dominant role for monocytic cells in progression.

BACKGROUND: Prostate cancer is caused by genomic aberrations in normal epithelial cells, however clinical translation of findings from analyses of cancer cells alone has been very limited. A deeper understanding of the tumour microenvironment is needed to identify the key drivers of disease progression and reveal novel therapeutic opportunities. RESULTS: In this study, the experimental enrichment of selected cell-types, the development of a Bayesian inference model for continuous differential transcript abundance, and multiplex immunohistochemistry permitted us to define the transcriptional landscape of the prostate cancer microenvironment along the disease progression axis. An important role of monocytes and macrophages in prostate cancer progression and disease recurrence was uncovered, supported by both transcriptional landscape findings and by differential tissue composition analyses. These findings were corroborated and validated by spatial analyses at the single-cell level using multiplex immunohistochemistry. CONCLUSIONS: This study advances our knowledge concerning the role of monocyte-derived recruitment in primary prostate cancer, and supports their key role in disease progression, patient survival and prostate microenvironment immune modulation.

Introgression of ASIP and TYRP1 Alleles Explains Coat Color Variation in Valais Goats.

The Valais Blackneck goat is a Swiss goat breed with a characteristic coat color phenotype. Before the revision of the breed standard in 1938, 4 different color varieties of Valais goats were known. Besides Blackneck animals resembling the modern breed standard, the brown and white Copperneck goat, the white Capra Sempione, and the greyish Grüenochte comprised the historic Valais goats. The brown pigmentation of Copperneck goats had previously been traced back to an introgression of a mutant TYRP1 allele from Toggenburg goats. In the present study, we identified additional introgression events of distinct ASIP alleles causing the remaining 2 rare coat color patterns within the Valais Blackneck goat breed. We identified the introgression of the AWt allele from Appenzell or Saanen goats in white Capra Sempione goats. Similarly, introgression of the Apc allele from Peacock goats resulted in the greyish Grüenochte phenotype. These results demonstrate past hybridization events between breeds that are separated today. A perfect genotype-phenotype association in 393 Valais goats supported the causality of the genotyped variants for the different coat color phenotypes. Our study gives insights into the introgression of functionally relevant copy number variant (CNV) alleles controlling pigmentation between goat breeds with strikingly different coat color patterns.

Evaluation of quality and gene expression of goat embryos produced in vivo and in vitro after cryopreservation.

In the present study, we aimed to identify morphological and molecular changes of in vivo and in vitro-produced goat embryos submitted to cryopreservation. In vivo embryos were recovered by transcervical technique from superovulated goats, whereas in vitro produced embryos were produced from ovaries collected at a slaughterhouse. Embryos were frozen by two-steps slow freezing method, which is defined as freezing to -32 °C followed by transfer to liquid nitrogen. Morphological evaluation of embryos was carried out by assessing blastocoel re-expansion rate and the total number of blastomeres. The expression profile of candidate genes related to thermal and oxidative stress, apoptosis, epigenetic, and implantation control was measured using RT-qPCR based SYBR Green system. In silico analyses were performed to identify conserved genes in goat species and protein-protein interaction networks were created. In vivo-produced embryos showed greater blastocoel re-expansion and more blastomere cells (P < 0.05). The expression level of CTP2 and HSP90 genes from in vitro cryopreserved embryos was higher than their in vivo counterparts. Unlikely, no significant difference was observed in the transcription level of SOD gene between groups. The high similarity of CPT2 and HSP90 proteins to their orthologs among mammals indicates that they share conserved functions. In summary, cryopreservation negatively affects the morphology and viability of goat embryos produced in vitro and changes the CPT2 and HSP90 gene expression likely in response to the in vitro production process.