Multimodal distribution and its impact on the accurate assessment of spermatozoa morphological data: Lessons from machine learning.

Objective assessment of sperm morphology is an essential component for assessing ejaculate quality. Due to economic limitations, investigators often divert to conducting observational studies instead of experimental ones, which provide the strongest statistical power, yielding more heterogeneous data regardless of the number of data sources (barns/farms). Using such data inevitably leads to higher variances of estimates, which negatively impacts the statistical power of a study. In this article, we describe a statistical methodology called finite mixture modeling (FMM), which, based on the supplied data and assumed number of sub-classes, classifies the data into two or more homogeneous types of distributions and determines their fractional size relative to the entire cohort. The goal is to use statistical methods that will confound the variance of the sample. A figure from a previous publication was used to generate simulated data (n=1559) on the cytoplasmic droplet rate. We identified that a bi-modal distribution with two latent classes best described the simulated data. Post-hoc estimation showed that about 80 % of observations belonged to latent class 1, with 20 % in latent class 2. The FMM methodology identified a cutoff point of 8.7 %. Finally, when estimating the standard error for the total cohort, the FMM methodology yielded a 40 % reduction in the standard error compared to standard methodologies. In conclusion, here we show that FMM successfully confounded the variance of the data and, as such, yielded lower estimates of the variance than standard methodologies, increasing the statistical power of the cohort.

Contaminant toxicity of concern for boars and semen used in assisted reproduction programs.

The commercial swine industry utilizes artificial insemination (AI) in their breeding programs. With this assisted reproductive technology, the process starts by obtaining fresh ejaculates from desirable boars who are housed in a dedicated facility (i.e., stud) that also contains a clean-room laboratory where semen quality is assessed and then ejaculates processed into AI doses. In concert with AI adoption, disruptions in sow herd reproductive performance have been traced back to contributions made from the boar stud. Through field investigations and research, several extrinsic contaminants have been identified that impact semen quality either at the boar or AI-dose level. These contaminants can be categorized as either biological or chemical in origin, eliciting reprotoxic outcomes at the boar level and/or spermatotoxicity at the AI-dose level. Biological contaminants include multiple genera of primarily opportunistic microbes (i.e., bacteria, fungi), along with their secondary metabolites (e.g., endotoxins, exotoxins, mycotoxins). Chemical contaminants appear to originate from products used at the stud, and include cleaning agent/disinfectant residues, leachates from gloves and plastics, semen extender impurities, purified and drinking water impurities, and pesticides (i.e., biocides, fungicides, herbicides, insecticides, wood preservatives). In conclusion, contaminants are a real and constant threat to the health and productivity of a stud, and have caused significant reproductive and economic losses in the swine industry. The knowledge gained in recognizing the types and sources of contaminants provides a solid foundation for the development and implementation of pro-active strategies that mitigate risk to the industry.

Biological and chemical contaminants in extended porcine semen: Outcomes and diagnosis.

Animal studs that provide extended semen for breeding have a significant contribution to reproductive outcomes. This report highlights several biological and chemical contaminants in extended semen that were found to be the causative agent for disturbances to herd reproductive performance, along with the diagnostic approach used in contaminant identification. Biological contaminants of concern include bacteria, viruses, and molds. From our investigations, opportunistic bacteria of mammalian and purified water origin appeared to be the most common biologic contaminant in stud operations. Chemical contaminants were another major cause of disrupted herd subfertility. A variety of chemical contaminants with spermatoxic activities were identified, with their source being residual detergents and disinfectants, inferior semen extender (e.g., inclusion errors, impurities, inferior ingredients), reconstitution water quality, and plastic toxicity. Success in contaminant identification was best achieved through the combined use of objective pre-use data on the extended semen product along with post-use fecundity data from the breeding farm(s). Using a temporal overlayment and point of time determination, targeted in vitro diagnostics were employed, including spermiogram analyses, microbiological methodologies, and analytical chemistry. Investigation outcomes included establishing robust stud hygiene and sanitation procedures, implementation of scientifically-based quality assurance/quality control programs that use sperm-safe screening to validate quality and consistency of supplies prior to acceptance and use, and stud-specific input monitoring practices.

A multifaceted analytical approach for detecting effects on semen quality when using small sample sizes.

Driven by technical, logistical and economic limitations, detection of treatment effects on semen quality typically include the design and collection of small sample datasets. A consequence of these small sample studies is that they suffer low statistical power. Historically, researchers faced with small sample size studies have relied upon non-parametric analysis; however, this approach is still unlikely to tease out a true statistical significance based upon limited sample size. Here we propose a novel methodology that can be applied in small samples study situations that combines repeated measures ANOVA and Mixed-Effects linear regression models with Bayesian Linear regression modeling when evaluating for treatment effects on quantitative semen quality parameters. Using this methodology, we show that investigating the data with this multifaceted analytical technique results in improved reproducibility and sensitivity of the findings while minimizing the likelihood of Type 1 errors when combining the inference statistics from multiple models/methodologies using Bayes Factor analysis.

A PCR detection method for discerning Serratia marcescens in extended boar semen.

Serratia marcescens is a bacterial contaminant that can be spermicidal when present in extended boar semen that is typically stored prior to breeding use at 15 to 18 °C for several days. This particular contaminant appears to originate from carrier boars, where it resides in the preputial cavity, but has also been shown to then easily contaminate the semen-processing laboratory. Screening for carrier boars to date has been performed through detection of S.marcescens in ejaculates using traditional agar plate culture techniques. These agar growth techniques are labor and time consuming due to the need for sample °titration and temporal growth followed by isolation, leading to delays in identification. The aim of this study was to develop a rapid, sensitive traditional PCR technique that can detect the presence of S.marcescens in extended boar semen. Primers for the detection of S. marcescens 16S rRNA were designed and specificity tested. After PCR optimization, assay sensitivity was evaluated using extended boar semen that was inoculated with various physiological ratios of spermatozoa: S.marcescens (100:1, 50:1, 20:1, 10:1, 8:1, 6:1, 4:1, 2:1. 1:1 and 1:10). Samples, held at 16 °C, were tested every 24 h over a 96 h period, with bacterial DNA extraction performed at each time point using a commercial kit. As a final step, the developed technique was used to screen random samples of extended boar semen for S. marcescens contamination. Results showed that this PCR technique had a sensitivity (90%) and specificity (100%) at detecting S.marcescens in the different inoculated ratios as well as in random, naturally contaminated samples of extended boar semen. In conclusion, this study reports a traditional PCR technique that is effective at rapidly and accurately detecting the presence of S.marcescens in boar extended semen.

Pubertal age based on testicular and epididymal histology in Göttingen minipigs.

Göttingen minipigs are used worldwide as nonrodent animal models for toxicologic and pharmaceutical research. Having knowledge of the age for onset of puberty is an important consideration when designing such biomedical experiments. The present study reports an earlier age for puberty on the basis of changes in testicular spermatogenic development in the Göttingen minipig. Testes and epididymides of 24 Göttingen minipigs ranging 5 to 8 weeks of age were obtained for histologic observation after hematoxylin-eosin staining. Microscopic examination was performed to determine the prevalence of cell types (e.g., spermatogonia, primary and secondary spermatocytes, round spermatids, elongated spermatids, luminal sperm) in seminiferous tubules and for the presence of sperm in the cauda epididymis. Puberty was defined as having a majority cell type presence of elongated spermatids in the seminiferous tubules in conjunction with the presence of spermatozoa in the cauda epididymis. Puberty was identified in two males at 6 weeks of age, two males at 7 weeks of age, and in all 6 males at 8 weeks of age. In conclusion, the age of puberty in male Göttingen minipigs occurs by 8 weeks of age, an earlier age than previously reported.

The impact of bacteriospermia on boar sperm storage and reproductive performance.

Bacteriospermia is a documented risk to reproductive performance when using extended boar semen for artificial insemination. A substantial list of bacteria have been recovered from boar semen attributed to fecal, preputial, skin, and hair microorganisms, with these and other environmental bacteria from processing areas identified in doses prepared for artificial insemination. Gram-negative bacteria are most commonly recovered from extended doses, including both Enterobacteriaceae and environmental contaminants, such as those that inhabit water purification systems. The method of processing, distributing, and storing fresh liquid boar semen before insemination plays a role in bacterial growth dynamics and the degree to which the bacteria may damage the sperm or affect the sow. Not all bacterial isolates or contamination levels have the same impact on sperm, with multiple factors governing if and when storage longevity will be reduced through sperm-to-sperm agglutination, impaired motility, acrosome disruption, or loss of membrane viability. Suboptimal reproductive performance can occur because of reduced fertilizing capacity of the sperm or induction of a uterine environment hostile to sperm and/or embryonic survival. Effective bacterial control strategies are necessary to minimize the risk of bacteria contaminating extended semen doses, including monitoring programs designed for quick detection and intervention, should the need arise.

The protective effect of a 17°C holding time on boar sperm plasma membrane fluidity after exposure to 5°C.

The holding time (HT) is the period during which an ejaculate, either in a raw or diluted state, is held at 17°C before further processing for cold-storage. In boars, the HT positively influences select sperm quality parameters of semen cooled from 15 to 5°C, a range in temperature during which plasma membrane remodeling occurs. Objective insight into the effect of HT on plasma membrane organization remains unknown. Therefore, the present work sought to elucidate if HT contributes to minimizing alterations in boar sperm plasma membrane fluidity at the initial step of the cooling process in a cryopreservation practice (holding at 5°C) and in relation with select sperm quality parameters. Nineteen ejaculates from five boars were collected and processed according to different treatments: T1) Fresh diluted semen, 0h at 17°C; T2) Fresh diluted semen, 24h at 17°C (HT); T3) Sperm from T1 in a lactose-egg yolk (LEY) extender, 3h at 5°C; T4) Sperm from T2 in LEY, 3h at 5°C; T5) Sperm from T1 in LEY, 24h at 5°C; T6) Sperm from T2 in LEY, 24h at 5°C. Sperm motility was assessed using CASA, and sperm plasma membrane integrity and fluidity were evaluated by flow cytometry with dual labeling (M540/YO-PRO®-1). Results demonstrated that the lack of exposure to a HT (T5) results in reduced sample motility compared to those having a HT (T6), with sperm exposed to HT exhibiting less plasma membrane fluidity. Collectively, these results provide empirical evidence that incorporation of a HT in semen processing protects boar sperm against cold injury through maintenance of lipid architecture of the plasma membrane.

The potential risk of infectious disease dissemination via artificial insemination in swine.

Artificial insemination (AI) is one of the most widely used assisted reproductive technologies in swine. To maintain a healthy semen trade, it is crucial that diligence be given to managing and minimizing the chance of extended semen playing an epidemiological role in the transmission of infectious disease. In swine, pathogens of primary importance, which may be transmitted through semen include Aujeszky's disease, brucellosis, chlamydophilosis, porcine circovirus type 2, classical swine fever, Japanese encephalitis, leptospirosis, parvovirus, porcine reproductive and respiratory syndrome, rubulavirus, foot-and-mouth disease and swine vesicular disease. This paper will summarise the current state of knowledge pertaining to these pathogens in relation to swine AI.

Thermotemporal dynamics of contaminant bacteria and antimicrobials in extended porcine semen.

Bacterial contamination of extended porcine semen has been associated with deleterious effects on both semen quality and sow fertility. Retrospective, prospective and in vitro studies were performed to delineate the prevalence and behavior of certain bacterial contaminants in extended semen, and antimicrobial pharmacodynamics in various semen diluents. Retrospective review of extended semen samples submitted from North American boar studs for microbiological screening at the University of Pennsylvania Reference Andrology Laboratory in 2005 and 2006 yielded bacteriospermia prevalence rates of 17% (144/832) and 26% (256/984), respectively. In a prospective study of regional boar studs, of 91 extended semen samples tested over 1-y, 29% were positive for bacteriospermia. Retrospective and prospective studies both showed that the preponderance of contaminant positive samples occurred during the fall months (P<0.05). To better understand behavior of select contaminant bacteria, generation intervals were determined for Serratia marcescens (SM) and Achromobacter xylosoxidans (AX) at 16, 22 and 37 degrees C. Generation times were temperature-dependent, with intervals decreasing two- to four-fold as incubation temperature increased. Growth patterns for SM, AX and Burkholderia cepacia were evaluated in various semen diluents. The different diluents exhibited constant or episodic patterns of growth within and among bacteria throughout the 5-d test period. Kill-time kinetics at 37 degrees C of several genera of bacteria in four semen diluents containing amoxicillin, gentamicin, tylosin, and lincomycin/spectinomycin (single drug or combination) ranged from 75 to over 360min, and was highly dependent (P<0.05) upon both type of bacteria and semen diluent.

Sanitary procedures for the production of extended semen.

Semen is collected and processed from a variety of animal species for use in artificial insemination breeding programmes. Because of the inherent nature of the semen collection process, bacterial contamination of the ejaculate is a common occurrence. Additionally, manipulation of the ejaculate during processing in the laboratory can expose the sample to possible introduction of bacterial contamination. If preventative measures at the stud fail to adequately control these risks, decreases in semen quality, dose longevity and fertility may occur. Multiple mammalian and non-mammalian sources have been identified as origins of contamination in the stud. Knowledge of these sources has aided the industries in developing strategies that help in controlling the introduction of contaminant bacteria in extended semen. A primary step in minimizing contamination is in the practice of good hygiene by stud personnel. Prudent general sanitation protocols should also be followed in the laboratory, animal housing and semen collection areas. Cleanliness and attention to the actual semen collection process can also aid in reducing bacterial load originating from the stud semen donor. Attentiveness to all of these steps significantly contributes to an overall reduction in the type and amount of bacterial contamination. However, their complete elimination still remains unavoidable. To address residual bacteria load in the sample, antimicrobials are commonly used in semen extenders intended to promote in vitro sperm longevity beyond that of a few hours. Current research by the animal industries continues in the selection and prudent use of antimicrobials that will lead to the success and sustainability of this modality in controlling bacterial contamination.

An update on the use of B-mode ultrasonography in female pig reproduction.

After technological advances allowed for the adaptation of B-mode ultrasonography equipment for use in pig facilities, ultrasonography quickly established itself as an ideal diagnostic aid for determining pregnancy status in pigs. In recent years, B-mode ultrasonography has found increased application in its use for monitoring ovarian activity and in estimating time of ovulation in pigs. B-mode ultrasonography is also valuable in providing a detailed assessment of the sow's ovaries and uterus to determine if pathological conditions exist, which could be contributing to poor individual or herd reproductive performance. In its most recent application in pigs, the gilt genital tract has been characterized peripubertally by ultrasonography in order to detect onset of puberty. The purpose of this review is to provide an update on the current status of B-mode ultrasonography in pig reproduction, and how this technology can be of value when used in pig production medicine.

Single, transcervical insemination using frozen-thawed semen in the Greyhound: a case series study.

Transcervical insemination (TCI) has generated recent interest as an assisted reproductive technique in the dog. A case series study was performed to determine if TCI using frozen-thawed semen was a viable technique to offer in a general veterinary practice setting. Over a period exceeding 28 months, 137 Greyhound bitches were presented for assisted breeding. A single, timed insemination using a rigid cystoscope to aid in transcervical deposition of a frozen-thawed semen dose was given within 72 h after the behaviorally estrual bitch had a > 4 ng/mL serum progesterone concentration and estrus-categorized vaginal cytology. Litter size, pregnancy and whelping rate were collected; their association to semen center and stud dog were quantified. Of the 137 bitches, 117 were bred for one cycle and 20 were bred for two or more cycles, giving a total of 161 single, timed inseminations. Pregnancy rate was 89.4%, with 141 (87.5%) whelping. Litter size was 6.9+/-2.7 (mean+/-S.D.) pups. Semen center (P=0.84) and stud (P=0.79) had no effect on pregnancy. These results were quite favorable when compared to prior TCI studies, and are possibly due to the use of a single breed (i.e., Greyhound) with good fertility. This study supported the application of TCI, in Greyhounds, as a successful and viable service to offer in private practice. Additionally, these results have value in their use for benchmarking future breed-specific and TCI research. Serendipitously, the apparent fecundity results obtained in this observational study suggests a possible greater appreciation be given to breed composition and choice in assisted reproductive technique studies.

Determining sample size for the morphological assessment of sperm.

Morphologic assessment of spermatozoa is an integral component in the analysis of semen. Whether a technician rapidly screening semen quality at a commercial stud, a veterinarian performing breeding soundness examinations, a clinician at a reference andrology laboratory providing auditing or diagnostic services, or a researcher evaluating morphology as a part of a fertility study, it is important to make an informed decision regarding the number of spermatozoa to include in the morphology assessment. Application of basic statistical principles such as the nature of proportions, level of confidence in an observed value, and the interaction of sample size with precision, can and should be used in the decision process. This paper outlines in detail the application of these statistical principles in relation to the morphologic assessment of spermatozoa. Guidelines on how these principles can be utilized in practical situations are discussed. Additionally, methodologies for comparison of results within and between laboratories (an area easily prone to misinterpretation) are reviewed. It is hoped that through the use of these fundamental statistical principles, this paper will bring clarity and delineation to the science of quantifying the morphology of spermatozoa.

A technique for preserving retained distal cytoplasmic droplets in situ for immunofluorescence evaluation of ejaculated porcine spermatozoa.

Cytoplasmic droplets (CD) associated with mammalian sperm have traditionally been investigated after isolation from ejaculated sperm cells, or in fixed tissue sections from the epididymides. Many of the current techniques for preparing spermatozoa for immunofluorescence assay (IFA) induce separation of distal cytoplasmic droplets, particularly when membrane permeabilization is required. This article describes a technique capable of maintaining distal cytoplasmic droplets in situ on ejaculated porcine spermatozoa throughout the process of permeabilization and immunostaining. Key steps in this technique include fixation with 0.2%, glutaraldehyde, permeabilization with 0.05% TX-100, and microtube incubations for IFA. Utilizing glutaraldehyde fixation, this technique yielded a mean retention rate of 88% for distal cytoplasmic droplets on ejaculated porcine spermatozoa.

B-mode ultrasonographic evaluation of paired testicular diameter of mature boars in relation to average total sperm numbers.

Crossbred, meat-type terminal sire boars (n = 215) were randomly assigned by age group (240-300, 301-360, 361-420, 421-480, 481-540, 541-600, and >721 days). Stud boars were on a once or twice weekly semen collection schedule. Testis diameters, in duplicate, were obtained using B-mode ultrasonography. Summation of average left and right testis diameter within boar gave the paired testicular diameter (PTD). Average ejaculate volume, sperm concentration (sperm/ml), and total sperm numbers for each boar were determined using composite data (average values) obtained from the last four semen collections. There was a <0.5cm difference between left and right testis diameters, with the left testis being the larger of the two testes (P = 0.03). There was no difference in PTD found between age groups in this study. Conversely, a dramatic increase in average total sperm numbers (ATSN) was observed between boars of 240-300 days (57.0+/-27.4 x 10(9) sperm) and up to 420 days (118.6+/-33.6 x 10(9) sperm) of age. The ATSN (127+/-32.5 x 10(9) sperm) remained constant for the 421-480 to >721-day age groups. The correlation between PTD and ATSN was low (r = 0.24) in this study. The results of this study demonstrate that normal boars should exhibit <0.5cm diameter difference between testes. As observed in other studies, the left testis was usually larger than the right testis. Correlation of total sperm numbers in a boar ejaculate using a composite ejaculate score (average values) and PTD measurements obtained using B-mode ultrasonography was poor when used in boars >8 months of age.

B-mode ultrasonographic examination of the accessory sex glands of boars.

Thorough examinations of the reproductive system of boars are generally not performed on normal boars to be used for breeding; only boars with problems undergo a form of a breeding soundness examination. In order for veterinarians to identify pathological conditions, the normal architecture of the accessory sex glands needs to be described. The purpose of this study was to use B-mode ultrasonography to describe the accessory sex glands in the boar and to see if transrectal ultrasonography would be a viable option in which to obtain this data. Initially, cross-sectional saline bath examinations of accessory sex glands were performed on crossbred boar reproductive tracts (n = 4) using B-mode ultrasonography equipped with a 5 MHz dual frequency linear array transducer. In situ examinations were also performed on terminal line crossbred boars (n = 16) ranging in age from 10 to 23 months old using the same ultrasound methodology; four boars were under general anesthesia and the remaining 12 were standing in crates. Eight boars were abstinent for 2 days and the other eight had ejaculates collected 2 h prior to examination. The paired bulbourethral glands are best described as a long oval gland with a uniformly echogenic appearance with a large anechoic space in the center of the gland extending most of its length. The walls of the vesicular glands were found to be thin, with the parenchyma having multiple small echolucent areas that appeared to merge and form a central canal. The prostate gland was best identified as a pecan-sized gland with a uniform echogenic appearance. Visualization of the prostate gland was accomplished with more proficiency using the saline bath ultrasonography as compared to in situ examinations. All of the accessory sex glands could be examined using both methodologies of ultrasonographic examination with a 5 MHz frequency linear array transducer. It was determined that each accessory sex gland could be recognized, and differences between ejaculated and nonejaculated boars could be identified. The results of this study demonstrate that transrectal ultrasonography can be used as a diagnostic aid in assessing the accessory sex glands of boars.

Prostaglandin F2alpha added to extended boar semen at processing elicits in vitro myometrial contractility after 72 hours of storage.

Improved fertility will maximize productivity of the swine industry. Myometrial contractility is an essential component in the fertilization process because it is the mechanism by which spermatozoa are transported to the site of fertilization. In the present study, we evaluated the potential use of PGF2alpha supplementation to the extended pig semen in regard to inducing myometrial contractility of sows. Extended boar semen (80 mL) was supplemented with PGF2alpha (5 mg) for 72 h at 17 +/- 1 degrees C. Cumulative doses of 0.1, 1, 10 and 100 microL of the mixture were tested on uterine strips obtained from diestrus sows. An increase in myometrial contractility was recorded with PGF2alpha supplementation when compared to extended semen or extender treatment alone after 72 h of incubation. Addition of PGF2alpha to the extended boar semen at the time of the experiment did not differ from the 72 h treated group. The results from this study support that PGF2alpha preparations can be added to extended doses of boar semen at processing to enhance myometrial contractility at the time of insemination for up to 72 h.

Field investigations of bacterial contaminants and their effects on extended porcine semen.

Field investigations (n=23) were made over a 3-yr period at North American boar studs and farms in which the primary complaint was sperm agglutination in association with decreased sperm longevity of extended semen, and increased regular returns to estrus and/or vaginal discharges across parity. Microscopic examination of extended semen from these units revealed depressed gross motility (usually <30%), sperm agglutination, and sperm cell death occurring within 2 d of semen collection and processing regardless of the semen extender used. The extended semen exhibited a high number of induced acrosome abnormalities (>20%). Sample pH was acidic (5.7 to 6.4) in 93% of the submitted samples. Aerobic culture yielded a variety of bacteria from different genera. A single bacterial contaminant was obtained from 66% of the submitted samples (n=37 doses); 34% contained 2 or more different bacterial genera. The most frequently isolated contaminant bacteria from porcine extended semen were Alcaligenes xylosoxydans (n=3), Burkholderia cepacia (n=6), Enterobacter cloacae (n=6), Escherichia coli (n=6), Serratia marcescens (n=5), and Stenotrophomonas [Xanthomonas] maltophilia (n=6); these 6 bacteria accounted for 71% of all contaminated samples, and were spermicidal when re-inoculated and incubated in fresh, high quality extended semen. All contaminant bacteria were found to be resistant to the aminoglycoside gentamicin, a common preservative antibiotic used in commercial porcine semen extenders. Eleven genera were spermicidal in conjunction with an acidic environment, while 2 strains (E. coli, S. maltophilia) were spermicidal without this characteristic acidic environment. Bacteria originated from multiple sources at the stud/farm, and were of animal and nonanimal origin. A minimum contamination technique (MCT) protocol was developed to standardize hygiene and sanitation. This protocol focused on MCT's during boar preparation, semen collection, semen processing and laboratory sanitation. Implementation of the MCT, in addition to specific recommendations in stud management, resulted in the control of bacterial contamination in the extended semen.

Clinical and serologic evaluation of two llamas (Lama glama) infected with Toxoplasma gondii during gestation.

Two pregnant llamas (Lama glama) infected with Toxoplasma gondii and their offspring were evaluated clinically and serologically. Llama 1 was inoculated orally with 1,000 infective occysts of the P89 strain of T. gondii at 82 days of gestation (DOG). Llama 2 became naturally infected with T. gondii between 26 and 119 DOG. Both llamas remained clinically normal and delivered healthy offspring. Sera collected from both llamas during pregnancy and from their offspring before and after colostral ingestion were evaluated for antibodies to T. gondii by the modified agglutination test (MAT), latex agglutination test (LAT), indirect hemagglutination test (IHAT), and the Sabin-Feldman dye test (DT). In llama 1, MAT antibody titers were < 1:20, 1:320, 1:1,280, 1:640, and 1:80 at 82, 97, 109, 132, and 152 DOG, respectively. The MAT titers in naturally infected llama 2 were < 1:32, 1:320-1:640, and 1:1,280 at 26, 119-200, and 346 DOG, respectively. In both llamas, antibody titers in the DT were of similar magnitude as the MAT, but titers in the LAT and IHAT were inconsistent. Antibodies to T. gondii were not detected in precolostral sera obtained from offspring of both llamas suggesting there was no fetal T. gondii infection.