The major challenge for sheep producers in grazing production systems is the infections with gastrointestinal nematodes (GIN) (Mavrot
The main hair sheep breeds raised in the tropics of Mexico are Blackbelly, Pelibuey, Dorper, and Katahdin (Zaragoza-Vera
This study was conducted during the months of February to september 2021 at the Centre for Training and Reproduction of Minor Species (CECAREM) located in Villahermosa, Tabasco, Mexico (17°59′13″ North latitude and 92°55′10″ East longitude), with an altitude of 9 meters above sea level. The climate in the region is warm and humid, with an average temperature of 27 °C, relative humidity above 90 % and rainfall of 1,677.4 mm per year (CONAGUA, 2021).
The study include forty-seven adult Pelibuey ewes with an average age of 3.4 ± 0.76 years, average body condition score of 3.1 ± 0.4 and average live weight of 49.4 ± 9.1 kg were included. The ewe feeding system was based on grazing of
According to the reproductive management of the farms, controlled breeding was carried out for 30 days; ewes were synchronized using “male effect” (Alavez-Ramírez
The ewes were sampled weekly from the first week of gestation to the last week of weaning during the two physiological stages of evaluation (gestation and lactation). Five-ten grams of faeces were obtained directly from the rectum of each animal by means of polyethylene bags and were placed in coolers with ice for transport to the Tropical and Vector-Borne Diseases Laboratory, of the Juarez Autonomous University of Tabasco, Mexico. Samples were processed using the modified McMaster technique to determine the FEC (Rodríguez-Vivas & Cob-Galera, 2005) with sensitivity of 50 eggs/g−1. The genera of GIN involved in the infections were determined by coprocultures performed fortnightly from a pool of feces during the gestation and lactation periods following the methodology described by Corticelli & Lai (1963), and the identification was made as described by Van Wyk & Mayhew (2013). Blood samples were collected from each ewe by jugular vein venipuncture using tubes with EDTA (Vacutainer; BD Biosciences, Franklin Lakes, NJ, USA). Samples were used to determine the packed cell volume (PCV %), the number of eosinophils in peripheral blood (PEC, cells 103/μL), the plasma protein concentration (PP, g/dL) and the level of Immunoglobulin A (OD % against a positive control) against
During each visit to the CECAREM, the body condition (BCS) of each ewe was recorded using the Russel scale (1984).
The present study was approved by the Bioethics Committee of the Campus of Biological and Agricultural Sciences of the Autonomous University of Yucatan (CB-CCBA-D-2017-001). The samples were taken by expert Veterinarians according to the Mexican Official Standard guideline 051-Z00-1995 and the Mexican Official Standard to technical specifications for production, care and use of experimental animals.
The ewes were evaluated in the following period of gestation: early (1 – 4 weeks/February), mid (10 – 12 weeks/March) and late (20 – 21 weeks/June). During lactation period: early (1 – 3 weeks/July), mid (6 – 7 weeks/August) and late (9 – 11 weeks/September). For every stage, the mean and standard error (SE) of FEC were calculated. With these values, the cut-off point was determinate to classify ewes as susceptible to GIN (FEC values higher than mean + three SE), as resistant (FEC lower values than mean - three SE) and intermediate (FEC values between FEC > FEC - three SE and FEC < FEC + three SE) according to the methodology described by Morteo-Gómez
Statistical analysis of the productive period early lactation was carried out using the GLM procedure (SAS, 2004). The FEC was transformed into the logarithm [Log (FEC +1)] to reduce the variance and bring the model closer to a normal distribution. The statistical model was as follows:
Given that Yijkl = is the response variable (FEC, PCV), μ = Overall average, θi = Fixed effect type of birth (i=single, double), δ (γ)ij = Nested effect of study period on productive stage (i = 1–4, 10–12, 20–21 in gestation and 1–3, 6–7, 9–11 in lactation), ζ δ (γ)jk = Effect of animal type (Susceptible, intermediate and resistant) in each study period nested in productive stage, εijkl = Experimental error ~ IIDN (0, σ2). The comparison of means of each evaluated group was carried out with Tukey's test.
In order to know the validity of each productive stage, the following were calculated: the concordance value (Youden's J), sensitivity, specificity, positive and negative predictive values, the proportion of false positives and false negatives and the accuracy (Palomo-Couoh
With the FEC data of ewes resistant and not resistant a 2 × 2 table was constructed during the period of greatest FEC (early lactation) as a reference model and the periods in gestation and lactation was compared. In each period the ewes were determined as: a) True positives (TP): those resistant ewes that were resistant in the diagnostic stage of interest. False positives (FP): Ewes that were not resistant and were resistant in the evaluated diagnostic stage. True negatives (TN): non-resistant ewes that were negative (not resistant) in the diagnostic stage, and False negatives (FN): those resistant ewes that were negative (not resistant) in the diagnostic stage. With these data the Sensitivity = TP / (TP + FN), Specificity = TN / (FP + TN), Positive predictive value = TP / (TP + FP), Negative predictive value = TN / (FN + TN), Proportion of false positives = FP / (FP + TN) = 1 – Specificity, Proportion of false negatives = FN / (TP + FN) = 1 – Sensitivity, Accuracy = (TP + TN) / (TV + FP + FN + TN), Youden's J Index (Diagnostic Safety) = Sensitivity + Specificity – 1.
The Bioethics Committee of the Campus of Biological and Agricultural Sciences of the Autonomous University of Yucatan, Mexico, approved the present study for its development under authorization number CB-CCBA-D-2017-001.
The least mean squares of FEC of GIN during the different productive stages evaluated varied considerably. The highest FEC (Mean ± SE) was registered in the early lactation stage (weeks 1 – 3 of lactation, 1573 ± 386.81 SE) and the lower FEC was found in the early gestation stage (weeks 1 – 4 of gestation, 32 ± 25.06 SE), as seen in Table 1.
Predictive variables studied to identify resistance against GIN in pregnant and lactating Pelibuey ewes naturally infected during grazing under humid tropic conditions.
Reproductive stage | Weeks | N | FEC | PCV % | PEC cell 103/μl | PP g/dL | IgA % | BCS | ||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Mean | SE | Mean | SE | Mean | SE | Mean | SE | Mean | SE | Mean | SE | |||
Gestation | Early | 47 | 32.0 | 25.1 c | 30.2 | 0.6 b | 0.021 | 0.001 d | - | - | - | - | 3.1 | 0.1 a |
Mid | 166.9 | 71.4 c | 34.2 | 0.7 a | 0.023 | 0.001 d | 7.00 | 0.08 a | - | - | 3.0 | 0.0 ab | ||
Late | 518.0 | 139.8 b | 29.0 | 0.5 bc | 0.428 | 0.033 c | 6.86 | 0.07 a | 10.4 | 1.1 b | 3.0 | 0.0 ab | ||
Lactation | Early | 47 | 1572.8 | 386.6 a | 27.9 | 0.6 cd | 0.538 | 0.043 c | 6.57 | 0.08 b | 10.6 | 1.8 b | 3.0 | 0.0 ab |
Mid | 650.1 | 253.2 b | 26.4 | 0.7 de | 0.776 | 0.054 b | 6.59 | 0.08 b | 18.0 | 2.3 a | 2.9 | 0.1 b | ||
Late | 322.5 | 201.8 c | 25.5 | 0.6 c | 0.990 | 0.066 a | 6.37 | 0.08 b | 14.7 | 2.4 a | 2.7 | 0.1 c |
FEC: Fecal egg count; PCV: Packed Cell Volume; PEC: Peripheral Eosinophil Count; PP: Plasmatic Protein; IgA: Immunoglobulin A; BCS: Body Condition Score; SE: Standard Error. Different letter in the same column means statistical difference P<0.05; - values of PP and IgA not obtained in these periods.
The Figure 1 (n=47) shows the transformed values of FEC of GIN (Mean ± SE) according to ewe classification (susceptible, intermediate or resistant) during the different productive stages. In the productive stage early lactation, susceptible ewes had a mean FEC of 4369 ± 94.65 (21.73 % of the total ewe evaluated), the intermediate ewes group presented FEC of 1312 ± 33.86 (39.13 % of the total) and resistant ewes showed FEC of 353 ± 38.78 (39.13 % of the ewes evaluated).
The GIN genera identified from the larvae present in the coprocultures were
As can be seen in Table 1, PCV values decreased as the gestation and lactation periods progressed (P<0.05). Similar, PEC displayed a steady increase (P<0.05) during the study. PP levels decreased gradually as gestation progressed and showed lowest values during lactation periods (P<0.05).
IgA values showed differences according to the productive stage (late gestation-initial lactation and the rest of lactation) (P<0.05), highest values were obtained in mid and late lactation periods. A reduction in BCS was also evident during mid and late lactation periods (P<0.05).
About the type of birth, ewes with double birth showed higher means of FEC, PCV, and PP compared to single birth (P<0.05; Table 2). However, there were no differences to PEC, IGA, and BCS (P>0.05).
Predictive variables to classify Pelibuey ewes resistant against GIN according to the type of lambing under natural conditions of humid tropic of Mexico.
Variables | Single (n=20) | Double (n=27) | ||||
---|---|---|---|---|---|---|
N | Mean | SE | N | Mean | SE | |
FEC | 117 | 331 | 68 b | 161 | 605 | 95 a |
PCV % | 30.3 | 0.4 a | 28.0 | 0.4 b | ||
PEC cell 103/μl | 0.491 | 0.045 a | 0.437 | 0.035 a | ||
PP g/dL | 6.78 | 0.05 a | 6.59 | 0.05 b | ||
IgA % | 15.2 | 1.4 a | 12.0 | 1.0 a | ||
BCS | 3.0 | 0.0 a | 3.0 | 0.0 a |
FEC: Fecal egg count; PCV: Packed Cell Volume; PEC: Peripheral Eosinophil Count; PP: Plasmatic Protein; IgA: Immunoglobulin A; BCS: Body Condition Score; SE: Standard Error. Different letter in the same row means statistical difference P<0.05.
The results of concordance value (Youden's J), sensitivity, specificity, positive and negative predictive values, the proportion of false positives and false negatives, and the accuracy of values of each of the productive stages of evaluation to identify ewe susceptible or resistant to GIN infections are presented in Table 3. The higher sensitivity was observed in the gestation stage. However, specificity values showed a variation of 31 to 79 % and the predictive value of the positives showed a variation of 47 to 75 %. On the other hand, the early lactation stage was the reference model, and for this reason the values of sensitivity, specificity, predictive values of the positive and negative were 100 %. Unlike this stage, during the mean lactation, the sensitivity and predictive value of the negatives decreased as lactation progressed to the mid and late stages. The proportion of false positives ranged from 69 to 20 % during gestation with a gradual decrease reaching 0 % during early lactation. The proportion of false negatives was 0 % during the whole gestation and this value was maintained until the early lactation stage. Finally, the highest values of accuracy (100 %) and concordance (Youden's J = 1.0) were presented during early lactation.
Sensitivity, specificity, predictive positive and negative values, the proportion of positive and false, accuracy and J of Youden (concordance) of Pelibuey ewes classified susceptible or resistant against GIN during early, mid and late pregnancy and lactation periods.
Variable | Pregnancy | Lactation | ||||
---|---|---|---|---|---|---|
Early | Mid | Late | Early | Mid | Late | |
Sensitivity (Se) | 100 % | 100 % | 100 % | 100 % | 94.4 % | 72.2 % |
Specificity (Sp) | 31.0 % | 75.9 % | 79.3 % | 100 % | 100 % | 100 % |
Predictive Positive Value | 47.4 % | 72.0 % | 75.0 % | 100 % | 100 % | 100 % |
Predictive Negative Value | 100 % | 100 % | 100 % | 100 % | 96.7 % | 85.3 % |
Proportion of false positive | 69.0 % | 24.1 % | 20.7 % | 0 % | 0.0 % | 0 % |
Proportion of false negative | 0 % | 0 % | 0 % | 0 % | 5.6 % | 27.8 % |
Accuracy | 57.4 % | 85.1 % | 87.2 % | 100 % | 97.9 % | 89.4 % |
Youden's J | 0.3 | 0.8 | 0.8 | 1.0 | 0.9 | 0.7 |
The data were not transformed.
The selection of resistant sheep against GIN has been carried out mainly in lambs and to a lesser extent in adult ewes (González-Garduño
The FEC has been the most used parameter to classify susceptible and resistant ewes to GIN infections (Bouix
The variation on FEC showed along gestation to lactation stage is named “periparturient relaxation of immunity” (Gonzalez-Garduño
On the other hand, it has been observed that progesterone inhibits the development of
Concerning the present study results, during early lactation, the high FEC influenced by nutritional, hormonal, and immunological factors described above showed this productive stage as a period in which the challenge caused by GIN infections is ideal to select ewes.
The low FEC showed during the productive gestation phase could be explained by the nutrients in the ewe that maintain their immune capacity and control their parasite burden (Gonzalez-Garduño
Data for PCV and PP indicate a decrease in these values when comparing gestation with lactation. These results indicate the pathological effects of GIN infections on susceptible sheep. These parameters had a tendency to decrease as the lactation period progressed, which is consistent with reports about Morada Nova and Santa Inés ewe in Brazil (Bezerra
The concordance test performed in the present study showed that physiological stage to classify the ewes as resistant to GIN was the early lactation. The sensitivity, specificity, positive and negative predictive values, accuracy and the statistic of concordance Youden's J indicate that this stage is ideal for establishing ewe classification.
The evaluation of genetic resistance using molecular biology has contributed to identifying several genomic regions associated with decreased FEC. This suggests that parasite resistance involves a large number of genes contributing with small effects (Al Kalaldeh et al., 2019). Therefore, we consider that the proposed methodology in this study is currently more significant than molecular resistance due to involving different physiological stages and several field conditions that must be considered at the time of selection, instead of being limited to finding or not the presence of genes related to resistance. Moreover, we consider this methodology to be more easily applicable.
It is concluded that the optimal stage of production to evaluate phenotypic resistance against GIN infections in Pelibuey ewes was during the early lactation. This study demonstrated that during early lactation, the ewes showed the highest FEC of GIN. Low FEC of GIN during early and mid-gestation productive stages may lead to an erroneous selection of ewes that are not resistant to GIN. The number of lambs born and weaned is a factor that influences FEC independent from GIN resistance.